Fibre-type-specific and Mitochondrial Biomarkers of Muscle Damage after Mountain Races.

Consequences of running mountain races on muscle damage were investigated by analysing serum muscle enzymes and fibre-type-specific sarcomere proteins. We studied 10 trained amateur and 6 highly trained runners who ran a 35 km and 55 km mountain trail race (MTR), respectively. Levels of creatine kinase (CK), CK-MB isoform (CK-MB), sarcomeric mitochondrial CK (sMtCK), transaminases (AST and ALT), cardiac troponin I (cTnI) and fast (FM) and slow myosin (SM) isoforms, were assessed before, 1 h, 24 h and 48 h after the beginning of MTR. Significant SM increases were found at 24 h in the 55 km group. Levels of CK, CK-MB, AST and cTnI were significantly elevated in both groups following MTR, but in the 55 km group they tended to stabilize in at 48 h. Using pooled data, time-independent serum peaks of SM and CK-MB were significantly correlated. Moreover, concentration of sMtCK was significantly elevated at 1 and 24 h after the race in the 35 km group. Although training volume could confer protection on the mitochondria, the increase in serum CK-MB and SM in the 55 km group might be related to damage to the contractile apparatus type I fibres. Competing in long-distance MTRs might be related to deeper type I muscle fibre damage, even in highly trained individuals.

The MEF2 transcriptional target DMPK induces loss of sarcomere structure and cardiomyopathy.

Aims: The pathology of heart failure is characterized by poorly contracting and dilated ventricles. At the cellular level, this is associated with lengthening of individual cardiomyocytes and loss of sarcomeres. While it is known that the transcription factor myocyte enhancer factor-2 (MEF2) is involved in this cardiomyocyte remodelling, the underlying mechanism remains to be elucidated. Here, we aim to mechanistically link MEF2 target genes with loss of sarcomeres during cardiomyocyte remodelling. Methods and results: Neonatal rat cardiomyocytes overexpressing MEF2 elongated and lost their sarcomeric structure. We identified myotonic dystrophy protein kinase (DMPK) as direct MEF2 target gene involved in this process. Adenoviral overexpression of DMPK E, the isoform upregulated in heart failure, resulted in severe loss of sarcomeres in vitro, and transgenic mice overexpressing DMPK E displayed disruption of sarcomere structure and cardiomyopathy in vivo. Moreover, we found a decreased expression of sarcomeric genes following DMPK E gain-of-function. These genes are targets of the transcription factor serum response factor (SRF) and we found that DMPK E acts as inhibitor of SRF transcriptional activity. Conclusion: Our data indicate that MEF2-induced loss of sarcomeres is mediated by DMPK via a decrease in sarcomeric gene expression by interfering with SRF transcriptional activity. Together, these results demonstrate an unexpected role for DMPK as a direct mediator of adverse cardiomyocyte remodelling and heart failure.

Paracrine nitric oxide induces expression of cardiac sarcomeric proteins in adult progenitor cells through soluble guanylyl cyclase/cyclic-guanosine monophosphate and Wnt/ß-catenin inhibition.

AIM: Cardiac progenitor cells (CPC) from adult hearts can differentiate to several cell types composing the myocardium but the underlying molecular pathways are poorly characterized. We examined the role of paracrine nitric oxide (NO) in the specification of CPC to the cardiac lineage, particularly through its inhibition of the canonical Wnt/ß-catenin pathway, a critical step preceding cardiac differentiation. METHODS AND RESULTS: Sca1 + CPC from adult mouse hearts were isolated by magnetic-activated cell sorting and clonally expanded. Pharmacologic NO donors increased their expression of cardiac myocyte-specific sarcomeric proteins in a concentration and time-dependent manner. The optimal time window for NO efficacy coincided with up-regulation of CPC expression of Gucy1a3 (coding the alpha1 subunit of guanylyl cyclase). The effect of paracrine NO was reproduced in vitro upon co-culture of CPC with cardiac myocytes expressing a transgenic NOS3 (endothelial nitric oxide synthase) and in vivo upon injection of CPC in infarcted hearts from cardiac-specific NOS3 transgenic mice. In mono- and co-cultures, this effect was abrogated upon inhibition of soluble guanylyl cyclase or nitric oxide synthase, and was lost in CPC genetically deficient in Gucy1a3. Mechanistically, NO inhibits the constitutive activity of the canonical Wnt/ß-catenin in CPC and in cell reporter assays in a guanylyl cyclase-dependent fashion. This was paralleled with decreased expression of ß-catenin and down-regulation of Wnt target genes in CPC and abrogated in CPC with a stabilized, non-inhibitable ß-catenin. CONCLUSIONS: Exogenous or paracrine sources of NO promote the specification towards the myocyte lineage and expression of cardiac sarcomeric proteins of adult CPC. This is contingent upon the expression and activity of the alpha1 subunit of guanylyl cyclase in CPC that is necessary for NO-mediated inhibition of the canonical Wnt/ß-catenin pathway.

PRKCE gene encoding protein kinase C-epsilon-Dual roles at sarcomeres and mitochondria in cardiomyocytes.

Protein kinase C-epsilon (PKCε) is an isoform of a large PKC family of enzymes that has a variety of functions in different cell types. Here we discuss two major roles of PKCε in cardiac muscle cells; specifically, its role in regulating cardiac muscle contraction via targeting the sarcomeric proteins, as well as modulating cardiac cell energy production and metabolism by targeting cardiac mitochondria. The importance of PKCε action is described within the context of intracellular localization, as substrate selectivity and specificity is achieved through spatiotemporal targeting of PKCε. Accordingly, the role of PKCε in regulating myocardial function in physiological and pathological states has been documented in both cardioprotection and cardiac hypertrophy.

A myosin activator improves actin assembly and sarcomere function of human-induced pluripotent stem cell-derived cardiomyocytes with a troponin T point mutation.

We have investigated cardiac myocytes derived from human-induced pluripotent stem cells (iPSC-CMs) from two normal control and two family members expressing a mutant cardiac troponin T (cTnT-R173W) linked to dilated cardiomyopathy (DCM). cTnT is a regulatory protein of the sarcomeric thin filament. The loss of this basic charge, which is strategically located to control tension, has consequences leading to progressive DCM. iPSC-CMs serve as a valuable platform for understanding clinically relevant mutations in sarcomeric proteins; however, there are important questions to be addressed with regard to myocyte adaptation that we model here by plating iPSC-CMs on softer substrates (100 kPa) to create a more physiologic environment during recovery and maturation of iPSC-CMs after thawing from cryopreservation. During the first week of culture of the iPSC-CMs, we have determined structural and functional characteristics as well as actin assembly dynamics. Shortening, actin content, and actin assembly dynamics were depressed in CMs from the severely affected mutant at 1 wk of culture, but by 2 wk differences were less apparent. Sarcomeric troponin and myosin isoform composition were fetal/neonatal. Furthermore, the troponin complex, reconstituted with wild-type cTnT or recombinant cTnT-R173W, depressed the entry of cross-bridges into the force-generating state, which can be reversed by the myosin activator omecamtiv mecarbil. Therapeutic doses of this drug increased both contractility and the content of F-actin in the mutant iPSC-CMs. Collectively, our data suggest the use of a myosin activation reagent to restore function within patient-specific iPSC-CMs may aid in understanding and treating this familial DCM.

Matrix metalloproteinase-2 in oncostatin M-induced sarcomere degeneration in cardiomyocytes.

Cardiomyocyte dedifferentiation may be an important source of proliferating cardiomyocytes facilitating cardiac repair. Cardiomyocyte dedifferentiation and proliferation induced by oncostatin-M (OSM) is characterized by sarcomere degeneration. However, the mechanism underlying sarcomere degeneration remains unclear. We hypothesized that this process may involve matrix metalloproteinase-2 (MMP-2), a key protease localized at the sarcomere in cardiomyocytes. We tested the hypothesis that MMP-2 is involved in the sarcomere degeneration that characterizes cardiomyocyte dedifferentiation. Confocal immunofluorescence and biochemical methods were used to explore the role of MMP-2 in OSM-induced dedifferentiation of neonatal rat ventricular myocytes (NRVM). OSM caused a concentration- and time-dependent loss of sarcomeric α-actinin and troponin-I in NRVM. Upon OSM-treatment, the mature sarcomere transformed to a phenotype resembling a less-developed sarcomere, i.e., loss of sarcomeric proteins and Z-disk transformed into disconnected Z bodies, characteristic of immature myofibrils. OSM dose dependently increased MMP-2 activity. Both the pan-MMP inhibitor GM6001 and the selective MMP-2 inhibitor ARP 100 prevented sarcomere degeneration induced by OSM treatment. OSM also induced NRVM cell cycling and increased methyl-thiazolyl-tetrazolium (MTT) staining, preventable by MMP inhibition. These results suggest that MMP-2 mediates sarcomere degeneration in OSM-induced cardiomyocyte dedifferentiation and thus potentially contributes to cardiomyocyte regeneration.

Sex dimorphisms of crossbridge cycling kinetics in transgenic hypertrophic cardiomyopathy mice.

Familial hypertrophic cardiomyopathy (HCM) is a disease of the sarcomere and may lead to hypertrophic, dilated, restrictive, and/or arrhythmogenic cardiomyopathy, congestive heart failure, or sudden cardiac death. We hypothesized that hearts from transgenic HCM mice harboring a mutant myosin heavy chain increase the energetic cost of contraction in a sex-specific manner. To do this, we assessed Ca(2+) sensitivity of tension and crossbridge kinetics in demembranated cardiac trabeculas from male and female wild-type (WT) and HCM hearts at an early time point (2 mo of age). We found a significant effect of sex on Ca(2+) sensitivity such that male, but not female, HCM mice displayed a decrease in Ca(2+) sensitivity compared with WT counterparts. The HCM transgene and sex significantly impacted the rate of force redevelopment by a rapid release-restretch protocol and tension cost by the ATPase-tension relationship. In each of these measures, HCM male trabeculas displayed a gain-of-function when compared with WT counterparts. In addition, cardiac remodeling measured by echocardiography, histology, morphometry, and posttranslational modifications demonstrated sex- and HCM-specific effects. In conclusion, female and male HCM mice display sex dimorphic crossbridge kinetics accompanied by sex- and HCM-dependent cardiac remodeling at the morphometric, histological, and cellular level.

Acute heart failure with cardiomyocyte atrophy induced in adult mice by ablation of cardiac myosin light chain kinase.

AIMS: Under pressure overload, initial adaptive hypertrophy of the heart is followed by cardiomyocyte elongation, reduced contractile force, and failure. The mechanisms governing the transition to failure are not fully understood. Pressure overload reduced cardiac myosin light chain kinase (cMLCK) by ∼80% within 1 week and persists. Knockdown of cMLCK in cardiomyocytes resulted in reduced cardiac contractility and sarcomere disorganization. Thus, we hypothesized that acute reduction of cMLCK may be causative for reduced contractility and cardiomyocyte remodelling during the transition from compensated to decompensated cardiac hypertrophy. METHODS AND RESULTS: To mimic acute cMLCK reduction in adult hearts, the floxed-Mylk3 gene that encodes cMLCK was inducibly ablated in Mylk3(flox/flox)/merCremer mice (Mylk3-KO), and compared with two control mice (Mylk3(flox/flox) and Mylk3(+/+)/merCremer) following tamoxifen injection (50 mg/kg/day, 2 consecutive days). In Mylk3-KO mice, reduction of cMLCK protein was evident by 4 days, with a decline to below the level of detection by 6 days. By 7 days, these mice exhibited heart failure, with reduction of fractional shortening compared with those in two control groups (19.8 vs. 28.0% and 27.7%). Severely convoluted cardiomyocytes with sarcomeric disorganization, wavy fibres, and cell death were demonstrated in Mylk3-KO mice. The cardiomyocytes were also unable to thicken adaptively to pressure overload. CONCLUSION: Our results, using a new mouse model mimicking an acute reduction of cMLCK, suggest that cMLCK plays a pivotal role in the transition from compensated to decompensated hypertrophy via sarcomeric disorganization.

The angiotensin receptor-associated protein Atrap is a stimulator of the cardiac Ca2+-ATPase SERCA2a.

AIMS: The angiotensin II type 1 receptor-associated protein (Atrap) is highly expressed in the heart, but its function in the heart is unknown. We hypothesized that cardiac Atrap may interact with proteins other than the AT1 receptor. METHODS AND RESULTS: To identify potential novel interacting partners of Atrap, pull-down assays were performed. Sequencing by MALDI-MS of the isolated complexes showed that Atrap interacts with the cardiac Ca(2+)-ATPase SERCA2a. The interaction between Atrap and SERCA2a was confirmed by co-immunoprecipitation and by surface plasmon resonance (SPR) spectroscopy. Atrap enhanced the SERCA-dependent Ca(2+) uptake in isolated SR membrane vesicles. Furthermore, sarcomere shortenings and [Ca(2+)]i transients (CaTs) were determined in ventricular myocytes isolated from Atrap-/- and wild-type (WT) mice. The amplitudes of CaTs and sarcomere shortenings were similar in Atrap-/- and WT myocytes. However, the CaT decay and sarcomere re-lengthening were prolonged in Atrap-/- myocytes. To further evaluate the functional relevance of the Atrap-SERCA2a interaction in vivo, left-ventricular function was assessed in WT and Atrap-/- mice. The heart rates (564 ± 10 b.p.m. vs. 560 ± 11 b.p.m.; P = 0.80) and ejection fractions (71.3 ± 1.3 vs. 72 ± 1.8%; P = 0.79) were similar in WT and Atrap-/- mice, respectively (n = 15 for each genotype). However, the maximum filling rate (dV/dtmax) was markedly decreased in Atrap-/- (725 ± 48 µL/s) compared with WT mice (1065 ± 122 µL/s; P = 0.01; n = 15). CONCLUSION: We identified Atrap as a novel regulatory protein of the cardiac Ca(2+)-ATPase SERCA2a. We suggest that Atrap enhances the activity of SERCA2a and, consequently, facilitates ventricular relaxation.

Dominant negative Ras attenuates pathological ventricular remodeling in pressure overload cardiac hypertrophy.

The importance of the oncogene Ras in cardiac hypertrophy is well appreciated. The hypertrophic effects of the constitutively active mutant Ras-Val12 are revealed by clinical syndromes due to the Ras mutations and experimental studies. We examined the possible anti-hypertrophic effect of Ras inhibition in vitro using rat neonatal cardiomyocytes (NRCM) and in vivo in the setting of pressure-overload left ventricular (LV) hypertrophy (POH) in rats. Ras functions were modulated via adenovirus directed gene transfer of active mutant Ras-Val12 or dominant negative mutant N17-DN-Ras (DN-Ras). Ras-Val12 expression in vitro activates NFAT resulting in pro-hypertrophic and cardio-toxic effects on NRCM beating and Z-line organization. In contrast, the DN-Ras was antihypertrophic on NRCM, inhibited NFAT and exerted cardio-protective effects attested by preserved NRCM beating and Z line structure. Additional experiments with silencing H-Ras gene strategy corroborated the antihypertrophic effects of siRNA-H-Ras on NRCM. In vivo, with the POH model, both Ras mutants were associated with similar hypertrophy two weeks after simultaneous induction of POH and Ras-mutant gene transfer. However, LV diameters were higher and LV fractional shortening lower in the Ras-Val12 group compared to control and DN-Ras. Moreover, DN-Ras reduced the cross-sectional area of cardiomyocytes in vivo, and decreased the expression of markers of pathologic cardiac hypertrophy. In isolated adult cardiomyocytes after 2 weeks of POH and Ras-mutant gene transfer, DN-Ras improved sarcomere shortening and calcium transients compared to Ras-Val12. Overall, DN-Ras promotes a more physiological form of hypertrophy, suggesting an interesting therapeutic target for pathological cardiac hypertrophy.

Decreased creatine kinase is linked to diastolic dysfunction in rats with right heart failure induced by pulmonary artery hypertension.

Our objective was to investigate the role of creatine kinase in the contractile dysfunction of right ventricular failure caused by pulmonary artery hypertension. Pulmonary artery hypertension and right ventricular failure were induced in rats by monocrotaline and compared to saline-injected control animals. In vivo right ventricular diastolic pressure-volume relationships were measured in anesthetized animals; diastolic force-length relationships in single enzymatically dissociated myocytes and myocardial creatine kinase levels by Western blot. We observed diastolic dysfunction in right ventricular failure indicated by significantly steeper diastolic pressure-volume relationships in vivo and diastolic force-length relationships in single myocytes. There was a significant reduction in creatine kinase protein expression in failing right ventricle. Dysfunction also manifested as a shorter diastolic sarcomere length in failing myocytes. This was associated with a Ca(2+)-independent mechanism that was sensitive to cross-bridge cycling inhibition. In saponin-skinned failing myocytes, addition of exogenous creatine kinase significantly lengthened sarcomeres, while in intact healthy myocytes, inhibition of creatine kinase significantly shortened sarcomeres. Creatine kinase inhibition also changed the relatively flat contraction amplitude-stimulation frequency relationship of healthy myocytes into a steeply negative, failing phenotype. Decreased creatine kinase expression leads to diastolic dysfunction. We propose that this is via local reduction in ATP:ADP ratio and thus to Ca(2+)-independent force production and diastolic sarcomere shortening. Creatine kinase inhibition also mimics a definitive characteristic of heart failure, the inability to respond to increased demand. Novel therapies for pulmonary artery hypertension are needed. Our data suggest that cardiac energetics would be a potential ventricular therapeutic target.

Removal of Abnormal Myofilament O-GlcNAcylation Restores Ca2+ Sensitivity in Diabetic Cardiac Muscle.

Contractile dysfunction and increased deposition of O-linked ß-N-acetyl-d-glucosamine (O-GlcNAc) in cardiac proteins are a hallmark of the diabetic heart. However, whether and how this posttranslational alteration contributes to lower cardiac function remains unclear. Using a refined ß-elimination/Michael addition with tandem mass tags (TMT)-labeling proteomic technique, we show that CpOGA, a bacterial analog of O-GlcNAcase (OGA) that cleaves O-GlcNAc in vivo, removes site-specific O-GlcNAcylation from myofilaments, restoring Ca(2+) sensitivity in streptozotocin (STZ) diabetic cardiac muscles. We report that in control rat hearts, O-GlcNAc and O-GlcNAc transferase (OGT) are mainly localized at the Z-line, whereas OGA is at the A-band. Conversely, in diabetic hearts O-GlcNAc levels are increased and OGT and OGA delocalized. Consistent changes were found in human diabetic hearts. STZ diabetic hearts display increased physical interactions of OGA with α-actin, tropomyosin, and myosin light chain 1, along with reduced OGT and increased OGA activities. Our study is the first to reveal that specific removal of O-GlcNAcylation restores myofilament response to Ca(2+) in diabetic hearts and that altered O-GlcNAcylation is due to the subcellular redistribution of OGT and OGA rather than to changes in their overall activities. Thus, preventing sarcomeric OGT and OGA displacement represents a new possible strategy for treating diabetic cardiomyopathy.

Histone Deacetylase 3 (HDAC3)-dependent Reversible Lysine Acetylation of Cardiac Myosin Heavy Chain Isoforms Modulates Their Enzymatic and Motor Activity.

Reversible lysine acetylation is a widespread post-translational modification controlling the activity of proteins in different subcellular compartments. We previously demonstrated that a class II histone deacetylase (HDAC), HDAC4, and a histone acetyltransferase, p300/CREB-binding protein-associated factor, associate with cardiac sarcomeres and that a class I and II HDAC inhibitor, trichostatin A, enhances contractile activity of myofilaments. In this study we show that a class I HDAC, HDAC3, is also present at cardiac sarcomeres. By immunohistochemical and electron microscopic analyses, we found that HDAC3 was localized to A-band of sarcomeres and capable of deacetylating myosin heavy chain (MHC) isoforms. The motor domains of both cardiac α- and ß-MHC isoforms were found to be reversibly acetylated. Biomechanical studies revealed that lysine acetylation significantly decreased the Km for the actin-activated ATPase activity of MHC isoforms. By in vitro motility assay, we found that lysine acetylation increased the actin-sliding velocity of α-myosin by 20% and ß-myosin by 36% compared with their respective non-acetylated isoforms. Moreover, myosin acetylation was found to be sensitive to cardiac stress. During induction of hypertrophy, myosin isoform acetylation increased progressively with duration of stress stimuli independently of isoform shift, suggesting that lysine acetylation of myosin could be an early response of myofilaments to increase contractile performance of the heart. These studies provide the first evidence for localization of HDAC3 at myofilaments and uncover a novel mechanism modulating the motor activity of cardiac MHC isoforms.

Recovery of myofilament function through reactivation of glycogen synthase kinase 3ß (GSK-3ß): mechanism for cardiac resynchronization therapy.

The burden of noncommunicable diseases (NCDs) is emerging as a major public health challenge for the world. NCDs are projected to account for around 75 % of global mortality by the year 2020. The most common and problematic of all noncommunicable conditions is heart disease. As such, the weight of evidence supporting the routine use of cardiac resynchronization therapy (CRT) as a fruitful treatment for patients with ventricular dyssynchrony and moderate to severe chronic systolic heart failure is now quite substantial. The recent study done by Kirk et al. adds to our understanding of the effects of CRT in treating heart failure patients. Whether the current observations and findings extend to the more common clinical situations remains to be determined.

Sex-dependent, zinc-induced dephosphorylation of phospholamban by tissue-nonspecific alkaline phosphatase in the cardiac sarcomere.

We have previously reported that Zn(2+) infused into the coronary arteries of isolated rat hearts leads to the potent dephosphorylation of phospholamban (PLB) as well as a noticeable but less potent dephosphorylation of the ryanodine receptor 2. We hypothesized in the present study that a Zn(2+)-activated phosphatase is located in the vicinity of the sarcoplasmic reticulum (SR) where PLB and ryanodine receptor 2 reside. We report here the novel finding of tissue-nonspecific alkaline phosphatase (TNAP), a zinc-dependent enzyme, localized to the SR in the cardiac sarcomere of mouse myocardium. TNAP activity was enhanced by injection of Zn acetate into a tail vein before harvesting the heart and imaged using electron microscopy of electron dense deposits indicative of the hydrolysis of exogenous ß-glycerophosphate. TNAP activity was observed localized to the ends of the Z-line corresponding to SR and was qualitatively more visible in myocardium of males compared with females. Correspondingly, PLB phosphorylation status was potently reduced in myocardium of males injected with Zn acetate, whereas there was no apparent effect of Zn acetate injection on PLB phosphorylation in females. Surprisingly, Western blot analysis of TNAP content suggested a significantly lower TNAP content in males compared with females. These data suggest that TNAP plays a role in governing the phosphorylation status of calcium handling proteins in the SR. Furthermore, the content and activity of TNAP are differentially regulated between the sexes and thus may account for some sex differences in cardiopathologies associated with calcium handling.

The mechanism of calcium-induced inhibition of muscle fructose 1,6-bisphosphatase and destabilization of glyconeogenic complex.

The mechanism by which calcium inhibits the activity of muscle fructose 1,6-bisphosphatase (FBPase) and destabilizes its interaction with aldolase, regulating glycogen synthesis from non-carbohydrates in skeletal muscle is poorly understood. In the current paper, we demonstrate evidence that Ca(2+) affects conformation of the catalytic loop 52-72 of muscle FBPase and inhibits its activity by competing with activatory divalent cations, e.g. Mg(2+) and Zn(2+). We also propose the molecular mechanism of Ca(2+)-induced destabilization of the aldolase-FBPase interaction, showing that aldolase associates with FBPase in its active form, i.e. with loop 52-72 in the engaged conformation, while Ca(2+) stabilizes the disengaged-like form of the loop.

Cardiomyopathy mutations in the tail of ß-cardiac myosin modify the coiled-coil structure and affect integration into thick filaments in muscle sarcomeres in adult cardiomyocytes.

It is unclear why mutations in the filament-forming tail of myosin heavy chain (MHC) cause hypertrophic or dilated cardiomyopathy as these mutations should not directly affect contraction. To investigate this, we first investigated the impact of five hypertrophic cardiomyopathy-causing (N1327K, E1356K, R1382W, E1555K, and R1768K) and one dilated cardiomyopathy-causing (R1500W) tail mutations on their ability to incorporate into muscle sarcomeres in vivo. We used adenoviral delivery to express full-length wild type or mutant enhanced GFP-MHC in isolated adult cardiomyocytes. Three mutations (N1327K, E1356K, and E1555K) reduced enhanced GFP-MHC incorporation into muscle sarcomeres, whereas the remainder had no effect. No mutations significantly affected contraction. Fluorescence recovery after photobleaching showed that fluorescence recovery for the mutation that incorporated least well (N1327K) was significantly faster than that of WT with half-times of 25.1 ± 1.8 and 32.2 ± 2.5 min (mean ± S.E.), respectively. Next, we determined the effects of each mutation on the helical properties of wild type and seven mutant peptides (7, 11, or 15 heptads long) from the myosin tail by circular dichroism. R1382W and E1768K slightly increased the α-helical nature of peptides. The remaining mutations reduced α-helical content, with N1327K showing the greatest reduction. Only peptides containing residues 1301-1329 were highly α-helical suggesting that this region helps in initiation of coiled coil. These results suggest that small effects of mutations on helicity translate into a reduced ability to incorporate into sarcomeres, which may elicit compensatory hypertrophy.

Lysine methyltransferase Smyd2 regulates Hsp90-mediated protection of the sarcomeric titin springs and cardiac function.

Protein lysine methylation controls gene expression and repair of deoxyribonucleic acid in the nucleus but also occurs in the cytoplasm, where the role of this posttranslational modification is less understood. Members of the Smyd protein family of lysine methyltransferases are particularly abundant in the cytoplasm, with Smyd1 and Smyd2 being most highly expressed in the heart and in skeletal muscles. Smyd1 is a crucial myogenic regulator with histone methyltransferase activity but also associates with myosin, which promotes sarcomere assembly. Smyd2 methylates histones and non-histone proteins, such as the tumor suppressors, p53 and retinoblastoma protein, RB. Smyd2 has an intriguing function in the cytoplasm of skeletal myocytes, where it methylates the chaperone Hsp90, thus promoting the interaction of a Smyd2-methyl-Hsp90 complex with the N2A-domain of titin. This complex protects the sarcomeric I-band region and myocyte organization. We briefly summarize some novel functions of Smyd family members, with a focus on Smyd2, and highlight their role in striated muscles and cytoplasmic actions. We then provide experimental evidence that Smyd2 is also important for cardiac function. In the cytoplasm of cardiomyocytes, Smyd2 was found to associate with the sarcomeric I-band region at the titin N2A-domain. Binding to N2A occurred in vitro and in yeast via N-terminal and extreme C-terminal regions of Smyd2. Smyd2-knockdown in zebrafish using an antisense oligonucleotide morpholino approach strongly impaired cardiac performance. We conclude that Smyd2 and presumably several other Smyd family members are lysine methyltransferases which have, next to their nuclear activity, specific regulatory functions in the cytoplasm of heart and skeletal muscle cells. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.

Overexpression of TNNI3K, a cardiac-specific MAPKKK, promotes cardiac dysfunction.

Cardiac troponin I-interacting kinase (TNNI3K) is a cardiac-specific kinase whose biological function remains largely unknown. We have recently shown that TNNI3K expression greatly accelerates cardiac dysfunction in mouse models of cardiomyopathy, indicating an important role in modulating disease progression. To further investigate TNNI3K kinase activity in vivo, we have generated transgenic mice expressing both wild-type and kinase-dead versions of the human TNNI3K protein. Importantly, we show that the increased TNNI3K kinase activity induces mouse cardiac remodeling, and its kinase activity promotes accelerated disease progression in a left-ventricular pressure overload model of mouse cardiomyopathy. Using an in vitro kinase assay and proteomics analysis, we show that TNNI3K is a dual-function kinase with Tyr and Ser/Thr kinase activity. TNNI3K expression induces a series of cellular and molecular changes, including a reduction of sarcomere length and changes in titin isoform composition, which are indicative of cardiac remodeling. Using antisera to TNNI3K, we show that TNNI3K protein is located at the sarcomere Z disc. These combined data suggest that TNNI3K mediates cell signaling to modulate cardiac response to stress.

Cytoskeletal role in protection of the failing heart by ß-adrenergic blockade.

Formation of a dense microtubule network that impedes cardiac contraction and intracellular transport occurs in severe pressure overload hypertrophy. This process is highly dynamic, since microtubule depolymerization causes striking improvement in contractile function. A molecular etiology for this cytoskeletal alteration has been defined in terms of type 1 and type 2A phosphatase-dependent site-specific dephosphorylation of the predominant myocardial microtubule-associated protein (MAP)4, which then decorates and stabilizes microtubules. This persistent phosphatase activation is dependent upon ongoing upstream activity of p21-activated kinase-1, or Pak1. Because cardiac ß-adrenergic activity is markedly and continuously increased in decompensated hypertrophy, and because ß-adrenergic activation of cardiac Pak1 and phosphatases has been demonstrated, we asked here whether the highly maladaptive cardiac microtubule phenotype seen in pathological hypertrophy is based on ß-adrenergic overdrive and thus could be reversed by ß-adrenergic blockade. The data in this study, which were designed to answer this question, show that such is the case; that is, ß(1)- (but not ß(2)-) adrenergic input activates this pathway, which consists of Pak1 activation, increased phosphatase activity, MAP4 dephosphorylation, and thus the stabilization of a dense microtubule network. These data were gathered in a feline model of severe right ventricular (RV) pressure overload hypertrophy in response to tight pulmonary artery banding (PAB) in which a stable, twofold increase in RV mass is reached by 2 wk after pressure overloading. After 2 wk of hypertrophy induction, these PAB cats during the following 2 wk either had no further treatment or had ß-adrenergic blockade. The pathological microtubule phenotype and the severe RV cellular contractile dysfunction otherwise seen in this model of RV hypertrophy (PAB No Treatment) was reversed in the treated (PAB ß-Blockade) cats. Thus these data provide both a specific etiology and a specific remedy for the abnormal microtubule network found in some forms of pathological cardiac hypertrophy.