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There is ample evidence that regular exercise exerts beneficial effects on left ventricular (LV) hypertrophy, remodeling and dysfunction produced by ischemic heart disease or systemic hypertension. In contrast, the effects of exercise on pathological LV hypertrophy and dysfunction produced by LV outflow obstruction have not been studied to date. Consequently, we evaluated the effects of 8 weeks of voluntary wheel running in mice (which mitigates post-infarct LV dysfunction) on LV hypertrophy and dysfunction produced by mild (mTAC) and severe (sTAC) transverse aortic constriction. mTAC produced similar to 40% LV hypertrophy and increased myocardial expression of hypertrophy marker genes but did not affect LV function, SERCA2a protein levels. apoptosis or capillary density. Exercise had no effect on global LV hypertrophy and function in mTAC but increased interstitial collagen, and ANP expression. sTAC produced similar to 80% LV hypertrophy and further increased ANP expression and interstitial fibrosis and, in contrast with mTAC, also produced LV dilation. systolic as well as diastolic dysfunction, pulmonary congestion, apoptosis and capillary rarefaction and decreased SERCA2a and ryanodine receptor (RyR) protein levels. LV diastolic dysfunction was likely aggravated by elevated passive isometric force and Ca(2+)-sensitivity of myofilaments. Exercise training failed to mitigate the sTAC-induced LV hypertrophy and capillary rarefaction or the decreases in SERCA2a and RyR. Exercise attenuated the sTAC-induced increase in passive isometric force but did not affect myofilament Ca(2+)-sensitivity and tended to aggravate interstitial fibrosis. In conclusion, exercise had no effect on LV function in compensated and decompensated cardiac hypertrophy produced by LV outflow obstruction, suggesting that the effect of exercise on pathologic LV hypertrophy and dysfunction depends critically on the underlying cause. (C) 2011 Elsevier Ltd. All rights reserved.

Despite the apparent appropriateness of left ventricular (LV) remodeling following myocardial infarction (MI), it poses an independent risk factor for development of heart failure. There is a paucity of studies into the molecular mechanisms of LV remodeling in large animal species. We took an unbiased molecular approach to identify candidate transcription factors (TFs) mediating the genetic reprogramming involved in post-MI LV remodeling in swine. Left ventricular tissue was collected from remote, non-infarcted myocardium, 3 weeks after MI-induction or sham-surgery. Microarray analysis identified 285 upregulated and 278 downregulated genes (FDR two-fold (p two-fold. Five TFs were identified in both TFBS and protein/DNA array analyses, which showed matching changes for COUP-TFII and glucocorticoid receptor (GR) only. Treatment of swine with the GR antagonist mifepristone after MI reduced the post-MI increase in LV mass, but LV dilation remained unaffected. Thus, using an unbiased approach to study post-MI LV remodeling in a physiologically relevant large animal model, we identified COUP-TFII and GR as potential key mediators of post-MI remodeling. Electronic supplementary material The online version of this article (doi:10.1007/s00395-011-0229-1) contains supplementary material, which is available to authorized users.

This review focuses on the current status of proteomic techniques that can be specifically applied to heart. Proteomics allows us to study alterations in protein expression in diseased hearts and leads us to develop new diagnostics and therapeutic parameters. The availability of the high resolution capacity of 2-DE can be successfully used to separate proteins in the first dimension according to their charge (isoelectric point) under denaturing conditions followed by their separation according to their molecular mass by SDS PAGE. The separated proteins are then visualized at high sensitivity with SYPRO dyes, especially SYPRO Ruby which is the most appropriate post-electrophoretic stain because of its compatibility for subsequent MS analysis. After the generation of a large protein dataset, they are organized using bioinformatics. Even though proteomics techniques have undergone substantial improvement, it remains a problem to identify phosphorylated proteins, which may be used for early disease detection. The proteomics analysis discussed in this review can be used for drug discovery, development of therapeutic modalities for cardiovascular diseases and the design of clinical trials. Proteins play more dynamic roles compared to DNA and RNA since most biological functions are regulated by protein-protein interactions. Protein-protein interaction mapping is crucial for many degenerative diseases and proteomics play an important role in understanding the molecular mechanisms of cellular functions. Though advancements in equipmentation have been made, it is unlikely to gain although MS is a powerful and evolving technique, the cost of running a sample needs to be considered. For example, regarding the cost of labeling, iTRAC runs about $400/sample and as many as 30 biological samples may be required to reach statistical significance in patient samples. Extensive time is also needed on a MS machine to run a fractionated sample on the order of days (times the number of samples). Once large datasets are generated, a bioinformaticist is required to align and analyze data from multiple treatment groups. An additional limitation is that the protein and splice variants have to be characterized to be identified by search engines. A number of predicted proteins may be identified with limited commercial resources available to follow up on such targets. Finally, though there have been advances in mass spectrometry equipment such as the Fouriertransform ion cyclotron resonance MS that generate higher sensitivity and dynamic range, there is a lack of standardization of protocols from sample collection and processing along the pipeline to data analysis. Unlike genomic data there is no community standard for database sharing. Although there are limitations to the technique, proteomics is likely to have great impact on drug discovery and clinical trial design leading to the development of niche personalized medicine. There is a definite need for early disease detection with appropriate biomarkers and proteomics are the tool to fulfill the requirement. For example, a routine, specific and sensitive serum proteomic pattern for cardiovascular diseases would be useful to clinicians for the early detection of diseases. In this regard, a low-resolution SELDI-TOF proteomic profile could be extremely useful. Compared to mRNAs, proteins are subjected to posttranslational modifications like phosphorylation, glycosylation and cleavage, and thus genomics are likely to miss the correct targets. This is of utmost importance for disease-related proteomics to become an essential component of personalized medicine system, which has great promise for the improvement of disease evaluation and patient care.

de Waard MC, van der Velden J, Boontje NM, Dekkers DH, van Haperen R, Kuster DW, Lamers JM, de Crom R, Duncker DJ. Detrimental effect of combined exercise training and eNOS overexpression on cardiac function after myocardial infarction. Am J Physiol Heart Circ Physiol 296: H1513-H1523, 2009; doi:10.1152/ajpheart.00485.2008.-It has been reported that exercise after myocardial infarction (MI) attenuates left ventricular (LV) pump dysfunction by normalization of myofilament function. This benefit could be due to an exercise-induced upregulation of endothelial nitric oxide synthase (eNOS) expression and activity. Consequently, we first tested the hypothesis that the effects of exercise after MI can be mimicked by elevated eNOS expression using transgenic mice with overexpression of human eNOS (eNOSTg). Both exercise and eNOSTg attenuated LV remodeling and dysfunction after MI in mice and improved cardiomyocyte maximal force development (F-max). However, only exercise training restored myofilament Ca2+-sensitivity and sarco(endo) plasmic reticulum Ca2+-ATPase (SERCA)2a protein levels and improved the first derivative of LV pressure at 30 mmHg. Conversely, only eNOSTg improved survival. In view of these partly complementary actions, we subsequently tested the hypothesis that combining exercise and eNOSTg would provide additional protection against LV remodeling and dysfunction after MI. Unexpectedly, the combination of exercise and eNOSTg abolished the beneficial effects on LV remodeling and dysfunction of either treatment alone. The latter was likely due to perturbations in Ca2+ homeostasis, as myofilament Fmax actually increased despite marked reductions in the phosphorylation status of several myofilament proteins, whereas the exercise-induced increases in SERCA2a protein levels were lost in eNOSTg mice. Antioxidant treatment with N-acetylcysteine or supplementation of tetrahydrobiopterin and L-arginine prevented these detrimental effects on LV function while partly restoring the phosphorylation status of myofilament proteins and further enhancing myofilament Fmax. In conclusion, the combination of exercise and elevated eNOS expression abolished the cardioprotective effects of either treatment alone after MI, which appeared to be, at least in part, the result of increased oxidative stress secondary to eNOS "uncoupling."

Diastolic dysfunction is general to all idiopathic dilated (IDCM) and hypertrophic cardiomyopathy (HCM) patients. Relaxation deficits may result from increased actin-myosin formation during diastole due to altered tropomyosin position, which blocks myosin binding to actin in the absence of Ca2+. We investigated whether ADP-stimulated force development (without Ca2+) can be used to reveal changes in actin-myosin blockade in human cardiomyopathy cardiomyocytes. Cardiac samples from HCM patients, harboring thick-filament (MYH7(mut), MYBPC3(mut)) and thin-filament (TNNT2(mut), TNNI3(mut)) mutations, and IDCM were compared with sarcomere mutation-negative HCM (HCMsmn) and nonfailing donors. Myofilament ADP sensitivity was higher in IDCM and HCM compared with donors, whereas it was lower for MYBPC3. Increased ADP sensitivity in IDCM, HCMsmn, and MYH7(mut) was caused by low phosphorylation of myofilament proteins, as it was normalized to donors by protein kinase A (PKA) treatment Troponin exchange experiments in a TNNT2(mut) sample corrected the abnormal actin-myosin blockade. In MYBPC3(trunc) samples, ADP sensitivity highly correlated with cardiac myosin-binding protein-C (cMyBP-C) protein level. Incubation of cardiomyocytes with cMyBP-C antibody against the actin-binding N-terminal region reduced ADP sensitivity, indicative of cMyBP-C's role in actin-myosin regulation. In the presence of Ca2+, ADP increased myofilament force development and sarcomere stiffness. Enhanced sarcomere stiffness in sarcomere mutation-positive HCM samples was irrespective of the phosphorylation background. In conclusion, ADP-stimulated contraction can be used as a tool to study how protein phosphorylation and mutant proteins alter accessibility of myosin binding on actin. In the presence of Ca2+, pathologic [ADP] and low PKA-phosphorylation, high actin-myosin formation could contribute to the impaired myocardial relaxation observed in cardiomyopathies.

Hypertrophic cardiomyopathy(HCM)results from mutations in genes encoding sarcomeric proteins, most often MYBPC3, which encodes cardiac myosin binding protein-C (cMyBP-C). A recently discovered HCM-associated 25-base pair deletion in MYBPC3 is inherited in millions worldwide. Although this mutation causes changes in the C10 domain of cMyBP-C (cMyBP-CC10mut), which binds to the light meromyosin (LMM) region of the myosin heavy chain, the underlying molecular mechanism causing HCM is unknown. In this study, adenoviral expression of cMyBP-CC10mut in cultured adult rat cardiomyocytes was used to investigate protein localization and evaluate contractile function and Ca2+ transients, compared with wildtype cMyBP-C expression (cMyBP-CWT) and controls. Fortyeight hours after infection, 44% of cMyBP-CWT and 36% of cMyBP-CC10mut protein levels were determined in total lysates, confirming equal expression. Immunofluorescence experiments showed little or no localization of cMyBP-CC10mut to the C-zone, whereas cMyBP-CWT mostly showed C-zone staining, suggesting that cMyBP-CC10mut could not properly integrate in the C-zone of the sarcomere. Subcellular fractionation confirmed that most cMyBP-CC10mut resided in the soluble fraction, with reduced presence in the myofilament fraction. Also, cMyBP-CC10mut displayed significantly reduced fractional shortening, sarcomere shortening, and relaxation velocities, apparently caused by defects in sarcomere function, because Ca2+ transients were unaffected. Co-sedimentation and protein cross-linking assays confirmed that C10mut causes the loss of C10 domain interaction with myosin LMM. Protein homology modeling studies showed significant structural perturbation in cMyBP-CC10mut, providing a potential structural basis for the alteration in its mode of interaction with myosin LMM. Therefore, expression of cMyBP-CC10mut protein is sufficient to cause contractile dysfunction in vitro.

Diastolic dysfunction in heart failure patients is evident from stiffening of the passive properties of the ventricular wall. Increased actomyosin interactions may significantly limit diastolic capacity, however, direct evidence is absent. From experiments at the cellular and whole organ level, in humans and rats, we show that actomyosin-related force development contributes significantly to high diastolic stiffness in environments where high ADP and increased diastolic [Ca(2+) ] are present, such as the failing myocardium. Our basal study provides a mechanical mechanism which may partly underlie diastolic dysfunction. Heart failure (HF) with diastolic dysfunction has been attributed to increased myocardial stiffness that limits proper filling of the ventricle. Altered cross-bridge interaction may significantly contribute to high diastolic stiffness, but this has not been shown thus far. Cross-bridge interactions are dependent on cytosolic [Ca(2+) ] and the regeneration of ATP from ADP. Depletion of myocardial energy reserve is a hallmark of HF leading to ADP accumulation and disturbed Ca(2+) handling. Here, we investigated if ADP elevation in concert with increased diastolic [Ca(2+) ] promotes diastolic cross-bridge formation and force generation and thereby increases diastolic stiffness. ADP dose-dependently increased force production in the absence of Ca(2+) in membrane-permeabilized cardiomyocytes from human hearts. Moreover, physiological levels of ADP increased actomyosin force generation in the presence of Ca(2+) both in human and rat membrane-permeabilized cardiomyocytes. Diastolic stress measured at physiological lattice spacing and 37°C in the presence of pathological levels of ADP and diastolic [Ca(2+) ] revealed a 76 ± 1% contribution of cross-bridge interaction to total diastolic stress in rat membrane-permeabilized cardiomyocytes. Inhibition of creatine kinase (CK), which increases cytosolic ADP, in enzyme-isolated intact rat cardiomyocytes impaired diastolic re-lengthening associated with diastolic Ca(2+) overload. In isolated Langendorff-perfused rat hearts, CK inhibition increased ventricular stiffness only in the presence of diastolic [Ca(2+) ]. We propose that elevations of intracellular ADP in specific types of cardiac disease, including those where myocardial energy reserve is limited, contribute to diastolic dysfunction by recruiting cross-bridges, even at low Ca(2+) , and thereby increase myocardial stiffness.

Differences in cardiac physiology are seen between men and women in terms of health and disease. Sex differences start to develop at puberty and are maintained during aging. The prevalence of almost all cardiovascular diseases is found to be higher in men than in women, and disease progression tends to be more rapid in male than in female patients. In cohorts of patients with hypertrophic cardiomyopathy (HCM), the most common autosomal inherited cardiac disease, men are overrepresented, suggesting increased penetrance of HCM-causing mutations in male patients. Cardiac remodeling in patients with HCM is higher in men than in women, the same is seen in HCM animal models. Patients with HCM are at increased risk of sudden cardiac death (SCD) and developing rhythm disorders. There seems to be no sex effect on the risk of SCD or arrhythmias in patients with HCM; however, animal studies suggest that certain mutations predispose men to SCD.

Rationale Cardiac myosin binding protein-C (cMyBP-C) is a trans-filament protein that has been shown to regulate cardiac function via its amino terminal (N′) region. In vitro studies have suggested the importance of the first 271 N′-residues of cMyBP-C (C0-C1f region) in slowing actin filament sliding over myosin to regulate cross-bridge cycling kinetics within the cardiac sarcomere. However, the role and necessity of the C0-C1f region of cMyBP-C in regulating contractile and cardiac function in vivo have not been elucidated. Hypothesis The N′-C0-C1f region of cMyBP-C is critical for proper cardiac function in vivo . Methods and Results Transgenic mice with approximately 95% expression of a mutant truncated cMyBP-C missing the N′-C0-C1f region (cMyBP-C 110 kDa ), compared to endogenous cMyBP-C, were generated and characterized at 3-months of age. cMyBP-C 110 kDa hearts had significantly elevated heart weight/body weight ratio, fibrosis, nuclear area and collagen content compared to hearts from non-transgenic (NTG) littermates. Electron microscopic analysis revealed normal sarcomere structure in cMyBP-C 110 kDa hearts but with apparently weaker cMyBP-C stripes. Furthermore, the ability of cMyBP-C to slow actin-filament sliding within the C-zone of native thick filaments isolated from NTG hearts was lost on thick filaments from cMyBP-C 110 kDa hearts. Short axis M-mode echocardiography revealed a significant increase in left ventricular (LV) internal diameter during diastole in cMyBP-C 110 kDa hearts. Importantly, cMyBP-C 110 kDa hearts displayed a significant reduction in fractional shortening compared to hearts from NTG mice. We further observed a decrease in the thickness of the LV interventricular septum and free wall during systole in cMyBP-C 110 kDa hearts. Strain analysis using images acquired from ECG-Gated Kilohertz Visualization identified a significant deficit in global longitudinal strain in cMyBP-C 110 kDa hearts compared to NTG hearts. Consistent with cardiac hypertrophy, we observed a significant increase in the expression of the hypertrophic genes MYH7 and NPPA by real-time PCR analysis. As expected, the expression levels of the MYBPC3 gene were significantly elevated in cMyBP-C 110 kDa hearts compared to NTG hearts. Surprisingly, our Western blot analyses revealed no significant difference in total cMyBP-C levels between NTG and cMyBP-C 110 kDa heart homogenates. However, intriguingly, we observed a significant elevation in cMyBP-C phosphorylation at Ser-273, Ser-282, and Ser-302, sites important for cMyBP-C9s regulation of actomyosin interaction, in cMyBP-C 110 kDa heart homogenates compared to those from NTG mice. Conclusion The N′-C0-C1f region of cMyBP-C is essential for maintaining normal cardiac morphology and function in vivo and loss of this region promotes contractile dysfunction both at the molecular and tissue level.

The giant myofilament protein titin has an extendable region that functions as a molecular spring. Cardiomyocytes have exquisite control over the length of titin, through splicing, enabling it to regulate passive stiffness. We hypothesized that titin as it sets the preload on the cardiomyocyte when stretched, together with diastolic Ca2+ pre-activates the cardiomyocyte during diastole and that this pre-activation is a major determinant for force production in the subsequent systolic phase. Via this route titin is thought to play an important role in active force development. Mutations in the splicing factor RNA binding motif protein (RBM20) results in the expression of large, highly compliant titin isoforms. In the present study we aimed to investigate the effect of long, highly compliant titin on the contractile properties of single cardiomyocytes. We measured single cardiomyocyte work-loops that mimic the cardiac cycle, in wildtype (WT) and heterozygous (HET) RBM20 deficient rats. In addition we studied detergent-permeabilized human patient samples that had known variations in titin based passive stiffness. At low pacing frequencies, myocytes isolated from HET left ventricles were unable to produce normal levels of work (55% of WT), but this difference disappeared when diastolic calcium increased at high pacing frequencies (>6 Hz). HET myocytes operated at higher SL to achieve the same level of work (2.1µm vs. 1.94µm at 6 Hz). In detergent-permeabilized cardiomyocytes isolated from human and rat heart we simulated cardiac twitches by transiently (0.5 s) exposing the cell to a physiological calcium concentration of pCa 5.7. Increasing pre-activation by bathing the cells in pCa 6.7 or pre-stretching the myocyte increased the kinetics of force development and thus the total force development within a transient activation. This is consistent with our hypothesis that pre-activation can increase force development in a time limited contraction such as a cardiac twitch. Pre-activation was pre-load dependent as the sarcomere length to which the myocytes had to be stretched for equivalent levels of pre-activation varied with the compliance of titin.

Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disorder. Mutations in the gene (MYBPC3) encoding cardiac myosin binding protein C (cMyBP-C) are a frequent cause of HCM. Clinical as well as animal-model studies have reported sex-related differences in HCM disease onset and severity. In addition, it has been established that physiological stimuli such as exercise may elicit a sexually dimorphic cardiac response. However, less attention has been paid to the sex-specific differences in the cellular pathophysiologic mechanisms underlying HCM. Therefore, we studied functional properties of the heart and sarcomeres in male and female sedentary and exercise (exposed to 8 weeks voluntary wheel running) mice.Echocardiography and isometric force measurements in mechanically isolated left ventricular (LV) membrane-permeabilized cardiomyocytes were performed in Wild-type (WT) and heterozygous (HET) knock-in mice carrying a Mybpc3 point mutation (G>A transition) associated with HCM.The LV mass was significantly lower in female WT and HET mice (23% in WT and 25% in HET), compared to corresponding male mice. Isometric force measurements revealed a significant lower maximal generated tension (Fmax) in HET male (13.0 ±1.1 kN/m2), than in females (20.0 ±2.2 kN/m2). Exercise induced a higher fractional shortening in HET male mice, which is correlated with an increased Fmax in exercised HET males. In contrast, LV weight was significantly increased in exercised HET females compared to sedentary females (7% in WT and 15% in HET). Ca2+-sensitivity was increased in exercised male and females WT mice. Similarly, Ca2+-sensitivity was enhanced in HET females, however not in exercised HET mice.In conclusion, exercise training improved cardiac and myofilament performance particularly in HET male mice, indicating that physiological stimuli may elicit a sexually dimorphic cardiac response in heterozygous Mybpc3-targeted knock-in mice.

Cardiac hypertrophy of the left ventricle (LV) in response to dynamic exercise-training (EX) is a beneficial adaptation to increased workload, and is thought to result from genetic reprogramming. We aimed to determine which transcription factors (TFs) are involved in this genetic reprogramming of the LV in swine induced by exercise-training. Swine underwent 3-6 weeks of dynamic EX, resulting in a 16% increase of LV weight/body weight ratio compared to sedentary animals (P = 0.03). Hemodynamic analysis showed an increased stroke volume index (stroke volume/body weight +35%; P = 0.02). Microarray-analysis of LV tissue identified 339 upregulated and 408 downregulated genes (false discovery rate < 0.05). Of the human homologues of the differentially expressed genes, promoter regions were searched for TF consensus binding sites (TFBSs). For upregulated and downregulated genes, 17 and 24 TFBSs were overrepresented by >1.5-fold (P< 0.01), respectively. In DNA-binding assays, using LV nuclear protein extracts and protein/DNA array, signal intensity changes >2-fold were observed for 23 TF-specific DNA probes. Matching results in TFBS and protein/ DNA array analyses were obtained for transcription factors YY1 (Yin Yang 1), PAX6 (paired box 6) and GR (glucocorticoid receptor). Notably, PAX6 and GR show lower signals in TFBS and protein/DNA array analyses upon exercise-training, whereas we previously showed higher signals for these factors in the remodeled LV of swine post-myocardial infarction (MI). In conclusion, we have identified transcription factors that may drive the genetic reprogramming underlying exercise-training induced LV hypertrophy in swine. PAX6 and GR are among the transcription factors that are oppositely regulated in LV hypertrophy after exercise-training and MI. These proteins may be at the base of the differences between pathological and physiological hypertrophy. (C) 2014 Elsevier Ltd. All rights reserved.

Cardiomyocyte size in the healthy heart is in part determined by the level of circulating thyroid hormone (TH). Higher levels of TH induce ventricular hypertrophy, primarily in response to an increase in hemodynamic load. Normal cardiac function is maintained in this form of hypertrophy, whereas progressive contractile dysfunction is a hallmark of pathological hypertrophy. MicroRNAs (miRNAs) are important modulators of signal-transduction pathways driving adverse remodeling. Because little is known about the involvement of miRNAs in cardiac TH action and hypertrophy, we examined the miRNA expression profile of the hypertrophied left ventricle (LV) using a mouse model of TH-induced cardiac hypertrophy. C57Bl/6J mice were rendered hypothyroid by treatment with propylthiouracil and were subsequently treated for 3 days with TH (T3) or saline. T3 treatment increased LV weight by 38% (p < 0.05). RNA was isolated from the LV and expression of 641 mouse miRNAs was determined using Taqman Megaplex arrays. Data were analyzed using RQ-manager and DataAssist. A total of 52 T3-regulated miRNAs showing a >2-fold change (p < 0.05) were included in Ingenuity Pathway Analysis to predict target mRNAs involved in cardiac hypertrophy. The analysis was further restricted to proteins that have been validated as key factors in hypertrophic signal transduction in mouse models of ventricular remodeling. A total of 27 mRNAs were identified as bona fide targets. The predicted regulation of 19% of these targets indicates enhancement of physiological hypertrophy, while 56% indicates suppression of pathological remodeling. Our data suggest that cardiac TH action includes a novel level of regulation in which a unique set of TH-dependent miRNAs primarily suppresses pathological hypertrophic signaling. This may be relevant for our understanding of the progression of adverse remodeling, since cardiac TH levels are known to decrease substantially in various forms of pathological hypertrophy.

Hypertrophic cardiomyopathy (HCM) is the most common form of inherited cardiac disease and the leading cause of sudden cardiac death in young people. HCM is caused by mutations in genes encoding contractile proteins. Cardiac myosin binding protein-C (cMyBP-C) is a thick filament contractile protein that regulates sarcomere organization and cardiac contractility. About 200 different mutations in the cMyBP-C gene (MYBPC3) have thus far been reported as causing HCM. Among them, a 25 base pair deletion in the branch point of intron 32 of MYBPC3 is widespread, particularly affecting people of South Asian descent, with 4% of this population carrying the mutation. This polymorphic mutation results in skipping of exon 33 and a reading frame shift, which, in turn, replaces the last 65 amino acids of the C-terminal C10 domain of cMyBP-C with a novel sequence of 58 residues (cMyBP-C(C10mut)). Carriers of the 25 base pair deletion mutation are at increased risk of developing cardiomyopathy and heart failure. Because of the high prevalence of this mutation in certain populations, genetic screening of at-risk groups might be beneficial. Scientifically, the functional consequences of C-terminal mutations and the precise mechanisms leading to HCM should be defined using induced pluripotent stem cells and engineered heart tissue in vitro or mouse models in vivo. Most importantly, therapeutic strategies that include pharmacology, gene repair, and gene therapy should be developed to prevent the adverse clinical effects of cMyBP-C(C10mut). This review article aims to examine the effects of cMyBP-C(C10mut) on cardiac function, emphasizing the need for the development of genetic testing and expanded therapeutic strategies.

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time. Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Hypertrophic cardiomyopathy (HCM) is predominantly caused by mutations in genes encoding sarcomeric proteins. One of the most frequent affected genes is MYBPC3, which encodes the thick filament protein cardiac myosin binding protein C. Despite the prevalence of HCM, disease pathology and clinical outcome of sarcomeric mutations are largely unknown. We hypothesized that microRNAs (miRNAs) could play a role in the disease process. To determine which miRNAs were changed in expression, miRNA arrays were performed on heart tissue from HCM patients with a MYBPC3 mutation (n = 6) and compared with hearts of non-failing donors (n = 6). 532 out of 664 analyzed miRNAs were expressed in at least one heart sample. 13 miRNAs were differentially expressed in HCM compared with donors (at p < 0.01, fold change >= 2). The genomic context of these differentially expressed miRNAs revealed that miR-204 (fold change 2.4 in HCM vs. donor) was located in an intron of the TRPM3 gene, encoding an aspecific cation channel involved in calcium entry. RT-PCR analysis revealed a trend towards TRPM3 upregulation in HCM compared with donor myocardium (fold change 23, p = 0.078). In silico identification of mRNA targets of differentially expressed miRNAs showed a large proportion of genes involved in cardiac hypertrophy and cardiac beta-adrenergic receptor signaling and we showed reduced phosphorylation of cardiac troponin I in the HCM myocardium when compared with donor. HCM patients with MYBPC3 mutations have a specific miRNA expression profile. Downstream mRNA targets reveal possible involvement in cardiac signaling pathways. (C) 2013 Elsevier Ltd. All rights reserved.

Rationale: High-myofilament Ca 2+ sensitivity has been proposed as a trigger of disease pathogenesis in familial hypertrophic cardiomyopathy (HCM) on the basis of in vitro and transgenic mice studies. However, myofilament Ca 2+ sensitivity depends on protein phosphorylation and muscle length, and at present, data in humans are scarce. Objective: To investigate whether high myofilament Ca 2+ sensitivity and perturbed length-dependent activation are characteristics for human HCM with mutations in thick and thin filament proteins. Methods and Results: Cardiac samples from patients with HCM harboring mutations in genes encoding thick ( MYH7 , MYBPC3 ) and thin ( TNNT2 , TNNI3 , TPM1 ) filament proteins were compared with sarcomere mutation-negative HCM and nonfailing donors. Cardiomyocyte force measurements showed higher myofilament Ca 2+ sensitivity in all HCM samples and low phosphorylation of protein kinase A (PKA) targets compared with donors. After exogenous PKA treatment, myofilament Ca 2+ sensitivity was similar ( MYBPC3 mut , TPM1 mut , sarcomere mutation-negative HCM), higher ( MYH7 mut , TNNT2 mut ), or even significantly lower ( TNNI3 mut ) compared with donors. Length-dependent activation was significantly smaller in all HCM than in donor samples. PKA treatment increased phosphorylation of PKA-targets in HCM myocardium and normalized length-dependent activation to donor values in sarcomere mutation-negative HCM and HCM with truncating MYBPC3 mutations but not in HCM with missense mutations. Replacement of mutant by wild-type troponin in TNNT2 mut and TNNI3 mut corrected length-dependent activation to donor values. Conclusions: High-myofilament Ca 2+ sensitivity is a common characteristic of human HCM and partly reflects hypophosphorylation of PKA targets compared with donors. Length-dependent sarcomere activation is perturbed by missense mutations, possibly via posttranslational modifications other than PKA hypophosphorylation or altered protein–protein interactions, and represents a common pathomechanism in HCM.

de Beer VJ, Taverne YJ, Kuster DW, Najafi A, Duncker DJ, Merkus D. Prostanoids suppress the coronary vasoconstrictor influence of endothelin after myocardial infarction. Am J Physiol Heart Circ Physiol 301: H1080-H1089, 2011. First published June 17, 2011; doi:10.1152/ajpheart.01307.2010.-Myocardial infarction (MI) is associated with endothelial dysfunction resulting in an imbalance in endothelium-derived vasodilators and vasoconstrictors. We have previously shown that despite increased endothelin (ET) plasma levels, the coronary vasoconstrictor effect of endogenous ET is abolished after MI. In normal swine, nitric oxide (NO) and prostanoids modulate the vasoconstrictor effect of ET. In light of the interaction among NO, prostanoids, and ET combined with endothelial dysfunction present after MI, we investigated this interaction in control of coronary vasomotor tone in the remote noninfarcted myocardium after MI. Studies were performed in chronically instrumented swine (18 normal swine; 13 swine with MI) at rest and during treadmill exercise. Furthermore, endothelial nitric oxide synthase (eNOS) and cyclooxygenase protein levels were measured in the anterior (noninfarcted) wall of six normal and six swine with MI. eNOS inhibition with N(omega)-nitro-L-arginine (L-NNA) and cyclooxygenase inhibition with indomethacin each resulted in coronary vasoconstriction at rest and during exercise, as evidenced by a decrease in coronary venous oxygen levels. The effect of L-NNA was slightly decreased in swine with MI, although eNOS expression was not altered. Conversely, in accordance with the unaltered expression of cyclooxygenase-1 after MI, the effect of indomethacin was similar in normal and MI swine. L-NNA enhanced the vasodilator effect of the ET(A/B) receptor blocker tezosentan but exclusively during exercise in both normal and MI swine. Interestingly, this effect of L-NNA was blunted in MI compared with normal swine. In contrast, whereas indomethacin increased the vasodilator effect of tezosentan only during exercise in normal swine, indomethacin unmasked a coronary vasodilator effect of tezosentan in MI swine both at rest and during exercise. In conclusion, the present study shows that endothelial control of the coronary vasculature is altered in post-MI remodeled myocardium. Thus the overall vasodilator influences of NO as well as its inhibition of the vasoconstrictor influence of ET on the coronary resistance vessels were reduced after MI. In contrast, while the overall prostanoid vasodilator influence was maintained, its inhibition of ET vasoconstrictor influences was enhanced in post-MI remote myocardium.

Protocols for the extraction of nuclear proteins have been developed for cultured cells and fresh tissue, but sometimes only frozen tissue is available. We have optimized the homogenization procedure and subsequent fractionation protocol for the preparation of nuclear protein extracts from frozen porcine left ventricular (LV) tissue. This method gave a highly reproducible protein yield (6.5 +/- 0.7% of total protein; mean +/- SE, n = 9) and a 6-fold enrichment of the nuclear marker protein B23. The nuclear protein extracts were essentially devoid of cytosolic, myofilament, and histone proteins. Compared to nuclear extracts from fresh LV tissue, some loss of nuclear proteins to the cytosolic fraction was observed. Using this method, we studied the distribution of tyrosine-phosphorylated signal transducer and activator of transcription 3 (PY-STAT3) in LV tissue of animals treated with the beta-agonist dobutamine. Upon treatment, PY-STAT3 increased 30.2 +/- 8.5-fold in total homogenates, but only 6.9 +/- 2.1-fold (n = 4, P = 0.03) in nuclear protein extracts. Of all PY-STAT3 formed, only a minor fraction appeared in the nuclear fraction. This simple and reproducible protocol yielded nuclear protein extracts that were highly enriched in nuclear proteins with almost complete removal of cytosolic and myofilament proteins. This nuclear protein extraction protocol is therefore well-suited for nuclear proteome analysis of frozen heart tissue collected in biobanks.

The fate of exogenous short-chain analogues of phosphatidylethanolamine and phosphatidylserine was studied in a deep-rough derivative of E. coli mutant strain AD93 that cannot synthesize phosphatidylethanolamine de novo. Using mass spectrometry, it was shown that dicaproyl(di 6:0)-phosphatidylethanolamine is extensively remodeled, eventually adopting the phosphatidylethanolamine species profile of the parental wild-type strain of AD93. Dicaproyl-phosphatidylserine was decarboxylated to form phosphatidylethanolamine, and yielded a species profile, which strongly resembled that of the introduced phosphatidylethanolamine. This demonstrates transport of phosphatidylserine to the cytosolic leaflet of the inner membrane. The changes of the species profile of phosphatidylethanolamine indicate that the short-chain phospholipids are most likely remodeled via two consecutive acyl chain substitutions, and at least part of this remodeling involves transport to the inner membrane.

During recent years it has become increasingly evident that cardiac myosin binding protein C (cMyBP-C) exerts an important role in regulation of sarcomere function with consequences for in vivo cardiac performance. The functional role of cMyBP-C is tightly regulated by kinase-mediated phosphorylation, of which protein kinase A (PKA)-mediated phosphorylation is important during cardiac stress and exercise. To date, three serines have been identified on which cMyBP-C can be phosphorylated in vivo, all of which are located in the cardiac isoform specific M region, i.e. Ser273, Ser282 and Ser 302. However, previous experiments have indicated that at least one more phosphorylation site should exist in humans. The aim of the present study was to identify the unknown phosphorylation site on human cMyBP-C. cMyBP-C was semi-purified from human non-failing donor and end-stage failing cardiac tissue. Tandem mass spectrometry was used to identify phosphorylation sites. Besides the already known Ser282 site, a phosphorylation site was detected on Ser133 in donor tissue. For characterization of this site, a phospho-specific antibody was developed. Levels of Ser133 phosphorylation are lower in end-stage heart failure samples as well as in samples from hypertrophic and dilated cardiomyopathy patients compared to donor. Initial experiments suggest that unlike the three previously known phosphorylation sites, Ser133 is not phosphorylated by PKA.In conclusion, we identified a fourth phosphorylation site (Ser133) in human cMyBP-C, which is present in the Pro-Ala rich region that links the C0 and C1 domains. This site is less phosphorylated in various cardiac pathologies and does not seem to be a target of PKA.Supported by the 7th Framework Program of the European Union (“BIG-HEART,” grant agreement 241577).

During recent years it has become increasingly evident that cardiac myosin binding protein C (cMyBP-C) exerts an important role in regulation of sarcomere function with consequences for in vivo cardiac performance. The functional role of cMyBP-C is tightly regulated by kinase-mediated phosphorylation. The most important kinase which phosphorylates cMyBP-C in vivo is protein kinase A (PKA), which is activated upon stimulation of the β-adrenergic receptors during exercise. In end-stage failing human myocardium, reduced phosphorylation of cMyBP-C has been reported using 1-dimensional and 2-dimensional gel electrophoresis. This reduced phosphorylation has been attributed to down-regulation and desensitization of the β-adrenergic receptor pathway in end-stage human heart failure. Low levels of cMyBP-C phosphorylation were also found in patients with familial hypertrophic cardiomyopathy (FHC), which is frequently caused by mutations in genes encoding sarcomeric proteins, with one exception: in FHC patients with mutations in the gene which encodes cMyBP-C (MYBPC3), phosphorylation of cMyBP-C was unaltered compared to non-failing donor hearts. cMyBP-C can be phosphorylated in vivo on at least three serine sites (Ser273, Ser282 and Ser 302), all of which are located in the cardiac isoform specific M region. At least one other site should exist in humans. With tandem mass spectrometry we recently identified a fourth phosphorylation site on Ser133, which is present in the Pro-Ala rich region that links the C0 and C1 domains. Preliminary data indicate that Ser133 is not phosphorylated by PKA. Analysis of phosphorylation on Ser133 in failing and donor samples revealed lower levels of Ser133 phosphorylation in several forms of cardiac disease compared to non-failing myocardium. Overall, our studies indicate that diverse cMyBP-C phosphorylation patterns exist in human cardiomyopathies, which may in part underlie sarcomere dysfunction observed in human heart failure.

Rationale: Myocardial infarction (MI) is associated with depressed cardiac contractile function and progression to heart failure. Cardiac myosin binding protein-C (cMyBP-C), a cardiac-specific myofilament protein, is proteolyzed post-MI in humans and results in an N-terminal fragment, C0C1f. The presence of C0C1f in cultured adult cardiomyocytes results in decreased Ca2+ transients and cell shortening, in addition to the induction of heart failure in a mouse model. However, the underlying mechanisms remain unclear.Objective: To determine how C0C1f causes altered contractility in human cardiac myofilaments in vitro.Methods and Results: We generated recombinant human C0C1f (hC0C1f) and incorporated it into skinned human left ventricular myocytes. Mechanical properties were then studied at sarcomere lengths of 2.0 and 2.3 µm. Our data demonstrate that the presence of hC0C1f in the sarcomere decreased maximal force myofilament Ca2+ sensitivity, increased cooperative activation at short lengths and enhanced length-dependent activation. Furthermore, hC0C1f led to increased cross-bridge cycling kinetics and tension cost at both short and long sarcomere lengths. We further established that the detrimental effects of hC0C1f occur through direct interaction with the thin filament proteins actin and α-tropomyosin (α-TM).Conclusions: Our data demonstrate that the presence of hC0C1f in the sarcomere is sufficient to induce depressed myofilament function and Ca2+ sensitivity in otherwise healthy human donor myofilament preparations. Decreased cardiac function post-MI may result, in part, from the ability of hC0C1f to bind actin and α-TM, suggesting that cleaved C0C1f could act as a poison peptide and disrupt the interaction of native cMyBP-C with the thin filament.Keywords: Cross-bridge cycling kinetics; length-dependent activation; cMyBP-C; C0C1f protein.