miR-1929-3p Overexpression Alleviates Murine Cytomegalovirus-Induced Hypertensive Myocardial Remodeling by Suppressing Ednra/NLRP3 Inflammasome Activation.

MicroRNAs (miRNAs) play crucial roles in the development of essential hypertension (EH). Previously, we found that the expression of miR-1929-3p was decreased in C57BL/6 mice with hypertension induced by murine cytomegalovirus (MCMV). In this study, we explored the role of miR-1929-3p in hypertension myocardial remodeling in MCMV-infected mice. First, we measured MCMV DNA and host IgG and IgM after infection and determined the expression of miR-1929-3p and its target gene endothelin A receptor (Ednra) mRNA in the myocardium of mice. Then, we performed invasive blood pressure (BP) monitoring. Heart-to-body weight ratio (HW/BW%), along with mRNA levels of B-type natriuretic peptide (BNP) and beta myosin heavy chain (ß-MHC), revealed myocardial remodeling. Hematoxylin/eosin and Masson's trichrome staining indicated morphological changes in the myocardium. Cardiac function was assessed via echocardiography. Moreover, MCMV-infected mice were injected with recombinant adeno-associated virus- (rAAV-) miR-1929-3p overexpression vector. Immunohistochemistry and western blotting showed the expression of Ednra and the activation of NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome. And enzyme-linked immunosorbent assay (ELISA) revealed the concentrations of endothelin-1 (ET-1), interleukin-1ß (IL-1ß), and interleukin-18 (IL-18). In this study, we found that decreased expression of miR-1929-3p in MCMV-infected mice induced high BP and further development of myocardial remodeling cardiac function injury through increased expression of Ednra. Strikingly, overexpression of miR-1929-3p ameliorated these pathological changes of the heart. The positive effect was shown to be associated with inhibition of NLRP3 inflammasome activation and decreased expression of key proinflammatory cytokine IL-1ß. Collectively, these results indicate that miR-1929-3p overexpression may effectively alleviate EH myocardial remodeling by suppressing Ednra/NLRP3 inflammasome activation in MCMV-infected mice.

Stiff matrix induces switch to pure ß-cardiac myosin heavy chain expression in human ESC-derived cardiomyocytes.

Human pluripotent stem cell (hPSC)-derived cardiomyocytes hold great potential for in vitro modeling of diseases like cardiomyopathies. Yet, knowledge about expression and functional impact of sarcomeric protein isoforms like the myosin heavy chain (MyHC) in hPSC-cardiomyocytes is scarce. We hypothesized that ventricular ß-MyHC expression alters contraction and calcium kinetics and drives morphological and electrophysiological differentiation towards ventricular-like cardiomyocytes. To address this, we (1) generated human embryonic stem cell-derived cardiomyocytes (hESC-CMs) that switched towards exclusive ß-MyHC, and (2) functionally and morphologically characterized these hESC-CMs at the single-cell level. MyHC-isoforms and functional properties were investigated during prolonged in vitro culture of cardiomyocytes in floating cardiac bodies (soft conditions) vs. culture on a stiff matrix. Using a specific anti-ß-MyHC and a newly generated anti-α-MyHC-antibody, we found individual cardiomyocytes grown in cardiac bodies to mostly express both α- and ß-MyHC-protein isoforms. Yet, 35 and 75 days of cultivation on laminin-coated glass switched 66 and 87 % of all cardiomyocytes to exclusively express ß-MyHC, respectively. Twitch contraction and calcium transients were faster for CMs on laminin-glass. Surprisingly, both parameters were only little affected by the MyHC-isoform, although hESC-CMs with only ß-MyHC had much lower ATP-turnover and tension cost, just as in human ventricular cardiomyocytes. Spontaneous contractions and no strict coupling of ß-MyHC to ventricular-like action potentials suggest that MyHC-isoform expression does not fully determine the hESC-CM differentiation status. Stiff substrate-induced pure ß-MyHC-protein expression in hESC-CMs, with several contractile parameters close to ventricular cardiomyocytes, provides a well-defined in vitro system for modeling of cardiomyopathies and drug screening approaches.

Exogenous apelin changes alpha and beta myosin heavy chain mRNA expression and improves cardiac function in PTU-induced hypothyroid rats.

The most important conditions associated with hypothyroidism is the cardiac dysfunction. Apelin is an endogenous ligand, involved in energy storage and metabolism which improves cardiac contractility. This study was done to evaluate the effects of apelin, l-Thyroxin (T4) or a combination of both, on cardiac function and mRNA expression of two contractile proteins, α and ß myosin heavy chain (α-MHC and ß-MHC), in 6-propyl-2-thiouracil (PTU)-induced hypothyroid rats. Forty male Wistar rats were randomly assigned into five groups: Ctrl (Control), and 4 hypothyroid groups (H, HA, HT, and HAT). The Hypothyroid (H) group received 0.05% PTU in the drinking water for six weeks; the next 3 groups, along with PTU, received apelin (HA, 200µg/kg/day, ip), T4 (HT, 20µg/kg/day, gavage), or a combination of both drugs (HAT) for the last 2weeks (weeks 5 and 6). TSH and T4 were measured using ELISA kit. Isolated hearts of animals were perfused in Langendorff apparatus and left ventricular developed pressure, cardiac contractility, heart rate, rate pressure product and perfusion pressure were assessed using PowerLab ADInstruments. In addition α-MHC and ß-MHC mRNA expression were evaluated by RT-PCR method in heart tissue. Apelin alone or accompanied by T4 significantly increased cardiac contractility and performance as compared to hypothyroid group. Apelin also significantly increased the alpha-MHC mRNA expression and in the presence of T4 significantly decreased beta-MHC mRNA expression. It seems that apelin alone may improve cardiac function in hypothyroid rats via genomic pathways.

Art27 interacts with GATA4, FOG2 and NKX2.5 and is a novel co-repressor of cardiac genes.

Transcription factors play a crucial role in regulation of cardiac biology. FOG-2 is indispensable in this setting, predominantly functioning through a physical interaction with GATA-4. This study aimed to identify novel co-regulators of FOG-2 to further elaborate on its inhibitory activity on GATA-4. The Art27 transcription factor was identified by a yeast-2-hybrid library screen to be a novel FOG-2 protein partner. Characterisation revealed that Art27 is co-expressed with FOG-2 and GATA-4 throughout cardiac myocyte differentiation and in multiple structures of the adult heart. Art27 physically interacts with GATA-4, FOG-2 and other cardiac transcription factors and by this means, down-regulates their activity on cardiac specific promoters α-myosin heavy chain, atrial natriuretic peptide and B-type natriuretic peptide. Regulation of endogenous cardiac genes by Art27 was shown using microarray analysis of P19CL6-Mlc2v-GFP cardiomyocytes. Together these results suggest that Art27 is a novel transcription factor that is involved in downregulation of cardiac specific genes by physically interacting and inhibiting the activity of crucial transcriptions factors involved in cardiac biology.

Nkx genes are essential for maintenance of ventricular identity.

Establishment of specific characteristics of each embryonic cardiac chamber is crucial for development of a fully functional adult heart. Despite the importance of defining and maintaining unique features in ventricular and atrial cardiomyocytes, the regulatory mechanisms guiding these processes are poorly understood. Here, we show that the homeodomain transcription factors Nkx2.5 and Nkx2.7 are necessary to sustain ventricular chamber attributes through repression of atrial chamber identity. Mutation of nkx2.5 in zebrafish yields embryos with diminutive ventricular and bulbous atrial chambers. These chamber deformities emerge gradually during development, with a severe collapse in the number of ventricular cardiomyocytes and an accumulation of excess atrial cardiomyocytes as the heart matures. Removal of nkx2.7 function from nkx2.5 mutants exacerbates the loss of ventricular cells and the gain of atrial cells. Moreover, in these Nkx-deficient embryos, expression of vmhc, a ventricular gene, fades, whereas expression of amhc, an atrial gene, expands. Cell-labeling experiments suggest that ventricular cardiomyocytes can transform into atrial cardiomyocytes in the absence of Nkx gene function. Through suggestion of transdifferentiation from ventricular to atrial fate, our data reveal a pivotal role for Nkx genes in maintaining ventricular identity and highlight remarkable plasticity in differentiated myocardium. Thus, our results are relevant to the etiologies of fetal and neonatal cardiac pathology and could direct future innovations in cardiac regenerative medicine.

Short-time glucose exposure of embryonic carcinoma cells impairs their function as terminally differentiated cardiomyocytes.

The fetal and postnatal phenotype is influenced by developmental conditions experienced prenatally. Among prenatal development metabolic factors are of particular importance as they are supposed to predispose for pathophysiological alterations later in life and to pioneer functional impairment in senescence (metabolic programming). Till now the mechanisms of metabolic programming are not well understood. We have investigated various concentrations of glucose during differentiation of pluripotent P19 embryonic carcinoma cells (ECC) into cardiomyocytes. Undifferentiated P19 cells were exposed to 5mM (low), 25 mM (control), 40 mM or 100mM (high) glucose for 48 h during embryoid body (EB) formation, followed by plating and differentiation into cardiomyocytes in vitro with standard glucose supplementation (25 mM) for 10-15 days. The amount of cardiac clusters, the frequency of spontaneous beatings as well as the expression of metabolic and cardiac marker genes and their promoter methylation were measured. We observed a metabolic programming effect of glucose during cardiac differentiation. Whereas the number of beating clusters and the expression of the cardiac marker alpha myosin heavy chain (α-MHC) were comparable in all groups, the frequencies of beating clusters were significantly higher in the high glucose group compared to low glucose. However, neither the insulin receptor (IR) or insulin like growth factor 1 receptor (IGF1R) nor the metabolic gene glucose transporter 4 (GLUT4) were influenced in RNA expression or in promoter methylation. Our data indicate that a short time glucose stress during embryonic cell determination leads to lasting effects in terminally differentiated cell function.

Cardiac and skeletal muscle expression of mutant ß-myosin heavy chains, degree of functional impairment and phenotypic heterogeneity in hypertrophic cardiomyopathy.

Several mutations in distinct genes, all coding for sarcomeric proteins, have been reported in unrelated kindreds with familial hypertrophic cardiomyopathy (FHC). We have identified nine individuals from three families harboring two distinct mutations in one copy of the ß-myosin heavy chain (ß-MHC) gene. In this study, the expression of the mutant ß-myosin protein isoform, isolated from slow-twitch fibers of skeletal muscle, was demonstrated by Northern and Western blot analysis; this myosin showed a decreased in vitro motility activity and produced a lower actin-activated ATPase activity. Isometric tension, measured in single slow-twitch fibers isolated from the affected individuals, also showed a significant decrease. The degree of impairment of ß-myosin function, as well as the loss in isometric tension development, were strictly dependent on the amount of the isoform transcribed from the mutated allele. Interestingly, a strong correlation was also demonstrated between mutant ß-myosin content and clinical features of FHC. On the other hand, we were unable to detect any correlation between mutant ß-myosin expression and degree of cardiac hypertrophy, thereby strengthening the hypothesis that hypertrophy, one of the hallmarks of FHC, might not necessarily be related to the clinical evolution of this disease. These findings lend support to the notion that additional factors rather than the mutated gene may play a pathogenetic role in cardiac wall thickening, whereas the prognosis appears to be strongly related to the amount of mutant protein.

Nox4-derived reactive oxygen species mediate cardiomyocyte injury in early type 1 diabetes.

Oxidative stress contributes to diabetic cardiomyopathy. This study explored the role of the NADPH oxidase Nox4 as a source of reactive oxygen species (ROS) involved in the development of diabetic cardiomyopathy. Phosphorothioated antisense (AS) or sense (S) oligonucleotides for Nox4 were administered for 2 wk to rats made diabetic by streptozotocin. NADPH oxidase activity, ROS generation, and the expression of Nox4, but Nox1 or Nox2, were increased in left ventricular tissue of the diabetic rats. Expression of molecular markers of hypertrophy and myofibrosis including fibronectin, collagen, α-smooth muscle actin, and ß-myosin heavy chain were also increased. These parameters were attenuated by the administration of AS but not S Nox4. Moreover, the impairment of contractility observed in diabetic rats was prevented in AS- but not S-treated animals. Exposure of cultured cardiac myocytes to 25 mM glucose [high glucose (HG)] increased NADPH oxidase activity, the expression of Nox4, and molecular markers of cardiac injury. These effects of HG were prevented in cells infected with adenoviral vector containing a dominant negative form of Nox4. This study provides strong evidence that Nox4 is an important source of ROS in the left ventricle and that Nox4-derived ROS contribute to cardiomyopathy at early stages of type 1 diabetes.

Resistin promotes cardiac hypertrophy via the AMP-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) and c-Jun N-terminal kinase/insulin receptor substrate 1 (JNK/IRS1) pathways.

Resistin has been suggested to be involved in the development of diabetes and insulin resistance. We recently reported that resistin is expressed in diabetic hearts and promotes cardiac hypertrophy; however, the mechanisms underlying this process are currently unknown. Therefore, we wanted to elucidate the mechanisms associated with resistin-induced cardiac hypertrophy and myocardial insulin resistance. Overexpression of resistin using adenoviral vector in neonatal rat ventricular myocytes was associated with inhibition of AMP-activated protein kinase (AMPK) activity, activation of tuberous sclerosis complex 2/mammalian target of rapamycin (mTOR) pathway, and increased cell size, [(3)H]leucine incorporation (i.e. protein synthesis) and mRNA expression of the hypertrophic marker genes, atrial natriuretic factor, brain natriuretic peptide, and ß-myosin heavy chain. Activation of AMPK with 5-aminoimidazole-4-carbozamide-1-ß-D-ribifuranoside or inhibition of mTOR with rapamycin or mTOR siRNA attenuated these resistin-induced changes. Furthermore, resistin increased serine phosphorylation of insulin receptor substrate (IRS1) through the activation of the apoptosis signal-regulating kinase 1/c-Jun N-terminal Kinase (JNK) pathway, a module known to stimulate insulin resistance. Inhibition of JNK (with JNK inhibitor SP600125 or using dominant-negative JNK) reduced serine 307 phosphorylation of IRS1. Resistin also stimulated the activation of p70(S6K), a downstream kinase target of mTOR, and increased phosphorylation of the IRS1 serine 636/639 residues, whereas treatment with rapamycin reduced the phosphorylation of these residues. Interestingly, these in vitro signaling pathways were also operative in vivo in ventricular tissues from adult rat hearts overexpressing resistin. These data demonstrate that resistin induces cardiac hypertrophy and myocardial insulin resistance, possibly via the AMPK/mTOR/p70(S6K) and apoptosis signal-regulating kinase 1/JNK/IRS1 pathways.

Adenovirus-mediated overexpression of cardiac troponin I-interacting kinase promotes cardiomyocyte hypertrophy.

1. Cardiac troponin I-interacting kinase (TNNI3K) is a novel cardiac-specific kinase gene. Quantitative real-time reverse transcription polymerase chain reaction analysis showed a significant increase in TNNI3K mRNA expression in hypertrophic cardiomyocytes induced by endothelin-1 (ET-1). The aim of the present study was to investigate the effects of TNNI3K on neonate rat cardiomyocyte hypertrophy induced by ET-1. 2. Adenoviruses were amplified in 293A cells. To determine a reasonable adenovirus infection dose cardiomyocytes were infected with an adenovirus carrying human TNNI3K (Ad-TNNI3K) at varying multiplicity of infection (MOI) and the expression of TNNI3K was analysed by western blot. 3. Cardiomyocytes were infected with either a control adenovirus carrying green fluorescent protein (Ad-GFP) or Ad-TNNI3K. Compared with Ad-GFP, the Ad-TNNI3K induced an increase in sarcomere organization, cell surface area, (3) H-leucine incorporation and ß-MHC re-expression. This type of hypertrophic phenomenon is similar to that observed in Ad-GFP-infected hypertrophic cardiomyocytes induced by ET-1. To determine the functional role of TNNI3K in ET-1-induced hypertrophic cardiomyocytes, the cells were infected with Ad-GFP or Ad-TNNI3K. Ad-TNNI3K induced an increase in sarcomere organization, cell surface area and (3) H-leucine incorporation compared with Ad-GFP. 4. These results suggest that TNNI3K overexpression induces cardiomyocytes hypertrophy and accelerates hypertrophy in hypertrophic cardiomyocytes. Therefore, TNNI3K might be an interesting target for the clinical treatment of hypertrophy.

Nuclear co-translocation of myotrophin and p65 stimulates myocyte growth. Regulation by myotrophin hairpin loops.

Myotrophin, a 12-kDa ankyrin repeat protein, stimulates protein synthesis and cardiomyocyte growth to initiate cardiac hypertrophy by activating the NF-kappaB signaling cascade. We found that, after internalization into myocytes, myotrophin cotranslocates into the nucleus with p65 to stimulate myocyte growth. We used structure-based mutations on the hairpin loops of myotrophin to determine the effect of the loops on myotrophin and p65 localization, induction of protein synthesis, and cardiac hypertrophy. Loop mutants, most prominently glutamic acid 33-->alanine (E33A), stimulated protein synthesis much less than wild type. Myotrophin-E33A internalized into myocytes but did not translocate into the nucleus and failed to promote nuclear translocation of p65. In addition, two cardiac hypertrophy marker genes, atrial natriuretic factor and beta-myosin heavy chain, were not up-regulated in E33A-treated cells. Myotrophin-induced myocyte growth and initiation of hypertrophy thus require nuclear co-translocation of myotrophin and p65, in a manner that depends crucially on the myotrophin hairpin loops.

Effect of mechanical loading on three-dimensional cultures of embryonic stem cell-derived cardiomyocytes.

Cardiomyocytes selected from murine embryonic stem cells (ESCs) using the cardiac-specific promoter alpha-myosin heavy chain were embedded into collagen and fibronectin scaffolds. A custom-built device was used to expose these constructs to mechanical loading (10% stretch at 1, 2, or 3 Hz) or no loading. Constructs were evaluated using reverse transcriptase polymerase chain reaction, histology, and immunohistochemistry. Mechanical loading significantly affected gene expression, and these changes were dependent on the frequency of stretch. A 1 Hz cyclical stretch resulted in significantly lower gene expression, whereas a 3 Hz cyclical stretch resulted in significantly greater gene expression than in unstretched controls. These constructs also developed cardiac-specific cell structures similar to those found in vivo. This study describes a 3-dimensional model to examine the direct effect of mechanical loading on the differentiation of ESC-derived cardiomyocytes embedded in a defined extracellular matrix scaffold. A technique was also developed to isolate the areas within the constructs undergoing the most homogeneous strain so that the effect of mechanical loading on gene expression could be directly evaluated. These experiments emphasize that ESC-derived cardiomyocytes are actively responding to cues from their environment and that those cues can drive phenotypic control and cardiomyocyte differentiation.

Fusion of bone marrow-derived stem cells with cardiomyocytes in a heterologous in vitro model.

OBJECTIVE: Recent studies have demonstrated that transplanted bone marrow-derived stem cells (BMCs) possess a broad differentiation potential and are able to form new cardiomyocytes. However, the identity of BMCs as true cardiomyocytes is still ambiguous. Therefore, we investigated the fate of transplanted fluorescence labeled BMCs and cardiomyocytes in co-culture. METHODS: For cell tracking we used two different fluorescent probes, Vybrant/DiO and Vybrant/DiI. BMCs were taken from human sternal marrow, purified using a Ficoll-gradient-centrifugation, treated with 5-azacytidine and stained with Vybrant/DiO. Furthermore, isolated spontaneous beating cardiomyocytes of neonatal rats (CM) were labeled with Vybrant/DiI. Thereafter, the BMCs were transplanted into CM-cultures and investigated on day 1, 4, 7, 14 and 28 using two-color fluorescence phenotyping by laser-scanning-cytometry (LSC). Two-color positive cells were harvested by patch-clamp technique and beta-MHC mRNA expression was analyzed by single-cell PCR. RESULTS: Two different morphological phenotypes were observed by LSC. First, isolated DiO labeled BMCs without contact or with direct cell contact to DiI labeled CMs. Second, some BMCs and CMs were double positive for DiO/DiI spontaneously forming hybrids. This population increased by 18% from day 1 to 4 and decreased only slightly until day 28. Additionally, few two-color positive cell formations expressed both human and rat specific beta-MHC mRNA as well as only human beta-MHC mRNA indicating that cell-fusion and transdifferentiation has occurred. CONCLUSION: These observations provide in vitro evidence for spontaneous cell fusion and transdifferentiation of BMCs in co-culture, raising the possibility that the observed phenomenons may contribute to development or maintenance of these cell types.

Developmental expression and comparative genomic analysis of Xenopus cardiac myosin heavy chain genes.

Myosin heavy chains (MHC) are cytoskeletal motor proteins essential to the process of muscle contraction. We have determined the complete sequences of the Xenopus cardiac MHC genes, alpha-MHC and ventricular MHC (vMHC), and have characterized their developmental expression profiles. Whereas alpha-MHC is expressed from the earliest stages of cardiac differentiation, vMHC transcripts are not detected until the heart has undergone chamber formation. Early expression of vMHC appears to mark the cardiac conduction system, but expression expands to include the ventricle and outflow tract myocardium during subsequent development. Sequence comparisons, transgenic expression analysis, and comparative genomic studies indicate that Xenopus alpha-MHC is the true orthologue of the mammalian alpha-MHC gene. On the other hand, we show that the Xenopus vMHC gene is most closely related to chicken ventricular MHC (vMHC1) not the mammalian beta-MHC. Comparative genomic analysis has allowed the detection of a mammalian MHC gene (MyH15) that appears to be the orthologue of vMHC, but evidence suggests that this gene is no longer active.

Physiology: postprandial cardiac hypertrophy in pythons.

Oxygen consumption by carnivorous reptiles increases enormously after they have eaten a large meal in order to meet metabolic demands, and this places an extra load on the cardiovascular system. Here we show that there is an extraordinarily rapid 40% increase in ventricular muscle mass in Burmese pythons (Python molurus) a mere 48 hours after feeding, which results from increased gene expression of muscle-contractile proteins. As this fully reversible hypertrophy occurs naturally, it could provide a useful model for investigating the mechanisms that lead to cardiac growth in other animals.

Morphological and functional alterations in ventricular myocytes from male transgenic mice with hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (FHC) is a human genetic disorder caused by mutations in sarcomeric proteins. It is generally characterized by cardiac hypertrophy, fibrosis, and myocyte disarray. A transgenic mouse model of FHC with mutations in the actin-binding domain of the alpha-myosin heavy chain (MyHC) gene displays many phenotypes similar to human FHC. At 4 months, male transgenic (TG) mice present with concentric cardiac hypertrophy that progresses to dilation with age. Accompanying this latter morphological change is systolic and diastolic dysfunction. Left ventricular (LV) myocytes from male TG and wild-type (WT) littermates at 5 and 12 months of age were isolated and used for morphological and functional studies. Myocytes from 5- and 12-month-old TG animals had shorter sarcomere lengths compared with WT. This sarcomere length difference was abolished in the presence of 2,3-butanedione monoxime, suggesting that the basal level of contractile element activation was increased in TG myocytes. Myocytes from 12-month-old TG mice were significantly longer than those from age-matched WT controls, and TG myocytes exhibited Z-band disorganization. When cells were paced at 0.5 Hz, TG myocyte relengthening and the fall in intracellular [Ca2+] were slowed when compared with cells from age-matched WT controls. Moreover, an increased amount of beta-myosin heavy chain protein was found in hearts from TG compared with WT. Thus, myocytes from the alpha-MyHC TG mouse model display many morphological and functional abnormalities that may help explain the LV dysfunction seen in this TG mouse model of FHC.

Heterologous expression of wild-type and mutant beta-cardiac myosin changes the contractile kinetics of cultured mouse myotubes.

The properties of myosin expressed in muscle are a major determinant of muscle performance. In this study we used a novel approach to examine the functional impact of changes in myosin heavy chain (MHC) isoform expression, as well as the consequences of expressing the mutant MHC implicated in familial hypertrophic cardiomyopathy (FHC). Cultured mouse myoblasts that normally express fast embryonic myosin were untransfected, or stably transfected with a plasmid expressing either wild-type (cWT) or mutant (D778G or G741R) beta-cardiac myosin. After differentiation for 5-7 days, cWT or mutant beta-cardiac myosin was expressed at 25 % of total myosin in the myotube. We measured time-to-peak shortening (ttp), time for half-relaxation (t0.5), the maximum velocity of shortening (Vmax) at 1 Hz stimulation, and the tetanic fusion frequency. Expression of cWT beta-cardiac myosin significantly increased ttp and t0.5 and decreased the fusion frequency compared with untransfected myotubes. However, when we compared myotubes expressing mutant beta-cardiac myosin with those expressing cWT beta-cardiac myosin, we found that ttp and t0.5 were significantly decreased, and Vmax was increased for the D778G mutant, whereas ttp, t0.5 and Vmax were unchanged for the G741R mutant. The fusion frequency was increased for both mutant myosins. Our data support the conclusion that the impact of the slower myosin isoform dominates when both slow and fast isoforms are present. This work suggests that FHC associated with either D778G or G741R mutation in MHC is an 'energy cost' disease, but that the phenotype of D778G is more severe than that of G741R.