Why is early-onset atrial fibrillation uncommon in patients with Duchenne Muscular Dystrophy? Insights from the mdx mouse.

BACKGROUND: A reduction in both dystrophin and neuronal nitric oxide synthase (NOS1) secondary to microRNA-31 (miR-31) upregulation contributes to the atrial electrical remodelling that underpins human and experimental atrial fibrillation (AF). By contrast, patients with Duchenne Muscular Dystrophy (DMD), who lack dystrophin and NOS1 and, at least in the skeletal muscle, have raised miR-31 expression, do not have increase susceptibility to AF in the absence of left ventricular (LV) dysfunction. Here we investigated whether dystrophin-deficiency is also associated with atrial upregulation of miR-31, loss of NOS1 protein, and increased AF susceptibility in young mdx mice. METHODS AND RESULTS: Echocardiography showed normal cardiac structure and function in 12- 13 weeks mdx mice, with no indication by assay of hydroxyproline that atrial fibrosis had developed. Absence of dystrophin in mdx mice was accompanied by an overall reduction in syntrophin and a lower NOS1 protein content in the skeletal muscle and in the left atrial and ventricular myocardium, with the latter occurring alongside reduced Nos1 transcript levels (exons 1-2 by qPCR) and an increase in NOS1-polyubiquitination (assessed using tandem polyubiquitination pulldowns; P<0.05 vs. WT). Neither the upregulation of miR-31 nor the substantial reduction in NOS activity observed in the skeletal muscle was present in the atrial tissue of mdx mice. At difference with the skeletal muscle, the mdx atrial myocardium showed a reduction in the constitutive NOS inhibitor, caveolin-1, coupled with an increase in NOS3 serine1177 phosphorylation, in the absence of differences in the protein content of other NOS isoforms or in the relative expression NOS1 splice variants. In line with these findings, transoesophageal atrial burst pacing revealed no difference in AF susceptibility between mdx mice and their wild type littermates. CONCLUSIONS: Dystrophin depletion is not associated with atrial miR-31 upregulation, reduced NOS activity or increased AF susceptibility in the mdx mouse. Compared with the skeletal muscle, the milder atrial biochemical phenotype may explain why patients with DMD do not exhibit a higher prevalence of atrial arrhythmias despite a reduction in NOS1 content.

Comparing the effects of chemical Ca2+ dyes and R-GECO on contractility and Ca2+ transients in adult and human iPSC cardiomyocytes.

We compared commonly used BAPTA-derived chemical Ca2+ dyes (fura2, Fluo-4, and Rhod-2) with a newer genetically encoded indicator (R-GECO) in single cell models of the heart. We assessed their performance and effects on cardiomyocyte contractility, determining fluorescent signal-to-noise ratios and sarcomere shortening in primary ventricular myocytes from adult mouse and guinea pig, and in human iPSC-derived cardiomyocytes. Chemical Ca2+ dyes displayed dose-dependent contractile impairment in all cell types, and we observed a negative correlation between contraction and fluorescence signal-to-noise ratio, particularly for fura2 and Fluo-4. R-GECO had no effect on sarcomere shortening. BAPTA-based dyes, but not R-GECO, inhibited in vitro acto-myosin ATPase activity. The presence of fura2 accentuated or diminished changes in contractility and Ca2+ handling caused by small molecule modulators of contractility and intracellular ionic homeostasis (mavacamten, levosimendan, and flecainide), but this was not observed when using R-GECO in adult guinea pig left ventricular cardiomyocytes. Ca2+ handling studies are necessary for cardiotoxicity assessments of small molecules intended for clinical use. Caution should be exercised when interpreting small molecule studies assessing contractile effects and Ca2+ transients derived from BAPTA-like chemical Ca2+ dyes in cellular assays, a common platform for cardiac toxicology testing and mechanistic investigation of cardiac disease physiology and treatment.

Endothelial cell-specific roles for tetrahydrobiopterin in myocardial function, cardiac hypertrophy, and response to myocardial ischemia-reperfusion injury.

The cofactor tetrahydrobiopterin (BH4) is a critical regulator of nitric oxide synthase (NOS) function and redox signaling, with reduced BH4 implicated in multiple cardiovascular disease states. In the myocardium, augmentation of BH4 levels can impact on cardiomyocyte function, preventing hypertrophy and heart failure. However, the specific role of endothelial cell BH4 biosynthesis in the coronary circulation and its role in cardiac function and the response to ischemia has yet to be elucidated. Endothelial cell-specific Gch1 knockout mice were generated by crossing Gch1fl/fl with Tie2cre mice, generating Gch1fl/flTie2cre mice and littermate controls. GTP cyclohydrolase protein and BH4 levels were reduced in heart tissues from Gch1fl/flTie2cre mice, localized to endothelial cells, with normal cardiomyocyte BH4. Deficiency in coronary endothelial cell BH4 led to NOS uncoupling, decreased NO bioactivity, and increased superoxide and hydrogen peroxide productions in the hearts of Gch1fl/flTie2cre mice. Under physiological conditions, loss of endothelial cell-specific BH4 led to mild cardiac hypertrophy in Gch1fl/flTie2cre hearts. Endothelial cell BH4 loss was also associated with increased neuronal NOS protein, loss of endothelial NOS protein, and increased phospholamban phosphorylation at serine-17 in cardiomyocytes. Loss of cardiac endothelial cell BH4 led to coronary vascular dysfunction, reduced functional recovery, and increased myocardial infarct size following ischemia-reperfusion injury. Taken together, these studies reveal a specific role for endothelial cell Gch1/BH4 biosynthesis in cardiac function and the response to cardiac ischemia-reperfusion injury. Targeting endothelial cell Gch1 and BH4 biosynthesis may provide a novel therapeutic target for the prevention and treatment of cardiac dysfunction and ischemia-reperfusion injury.NEW & NOTEWORTHY We demonstrate a critical role for endothelial cell Gch1/BH4 biosynthesis in coronary vascular function and cardiac function. Loss of cardiac endothelial cell BH4 leads to coronary vascular dysfunction, reduced functional recovery, and increased myocardial infarct size following ischemia/reperfusion injury. Targeting endothelial cell Gch1 and BH4 biosynthesis may provide a novel therapeutic target for the prevention and treatment of cardiac dysfunction, ischemia injury, and heart failure.

Proteomic Signature of Dysfunctional Circulating Endothelial Colony-Forming Cells of Young Adults.

Background A subpopulation of endothelial progenitor cells called endothelial colony-forming cells (ECFCs) may offer a platform for cellular assessment in clinical studies because of their remarkable angiogenic and expansion potentials in vitro. Despite endothelial cell function being influenced by cardiovascular risk factors, no studies have yet provided a comprehensive proteomic profile to distinguish functional (ie, more angiogenic and expansive cells) versus dysfunctional circulating ECFCs of young adults. The aim of this study was to provide a detailed proteomic comparison between functional and dysfunctional ECFCs. Methods and Results Peripheral blood ECFCs were isolated from 11 subjects (45% men, aged 27±5 years) using Ficoll density gradient centrifugation. ECFCs expressed endothelial and progenitor surface markers and displayed cobblestone-patterned morphology with clonal and angiogenic capacities in vitro. ECFCs were deemed dysfunctional if <1 closed tube formed during the in vitro tube formation assay and proliferation rate was <20%. Hierarchical functional clustering revealed distinct ECFC proteomic signatures between functional and dysfunctional ECFCs with changes in cellular mechanisms involved in exocytosis, vesicle transport, extracellular matrix organization, cell metabolism, and apoptosis. Targeted antiangiogenic proteins in dysfunctional ECFCs included SPARC (secreted protein acidic and rich in cysteine), CD36 (cluster of differentiation 36), LUM (lumican), and PTX3 (pentraxin-related protein PYX3). Conclusions Circulating ECFCs with impaired angiogenesis and expansion capacities have a distinct proteomic profile and significant phenotype changes compared with highly angiogenic endothelial cells. Impaired angiogenesis in dysfunctional ECFCs may underlie the link between endothelial dysfunction and cardiovascular disease risks in young adults.

Functional analysis of a gene-edited mouse model to gain insights into the disease mechanisms of a titin missense variant.

Titin truncating variants are a well-established cause of cardiomyopathy; however, the role of titin missense variants is less well understood. Here we describe the generation of a mouse model to investigate the underlying disease mechanism of a previously reported titin A178D missense variant identified in a family with non-compaction and dilated cardiomyopathy. Heterozygous and homozygous mice carrying the titin A178D missense variant were characterised in vivo by echocardiography. Heterozygous mice had no detectable phenotype at any time point investigated (up to 1 year). By contrast, homozygous mice developed dilated cardiomyopathy from 3 months. Chronic adrenergic stimulation aggravated the phenotype. Targeted transcript profiling revealed induction of the foetal gene programme and hypertrophic signalling pathways in homozygous mice, and these were confirmed at the protein level. Unsupervised proteomics identified downregulation of telethonin and four-and-a-half LIM domain 2, as well as the upregulation of heat shock proteins and myeloid leukaemia factor 1. Loss of telethonin from the cardiac Z-disc was accompanied by proteasomal degradation; however, unfolded telethonin accumulated in the cytoplasm, leading to a proteo-toxic response in the mice.We show that the titin A178D missense variant is pathogenic in homozygous mice, resulting in cardiomyopathy. We also provide evidence of the disease mechanism: because the titin A178D variant abolishes binding of telethonin, this leads to its abnormal cytoplasmic accumulation. Subsequent degradation of telethonin by the proteasome results in proteasomal overload, and activation of a proteo-toxic response. The latter appears to be a driving factor for the cardiomyopathy observed in the mouse model.

Inducibility, but not stability, of atrial fibrillation is increased by NOX2 overexpression in mice.

AIMS: Gp91-containing NADPH oxidases (NOX2) are a significant source of myocardial superoxide production. An increase in NOX2 activity accompanies atrial fibrillation (AF) induction and electrical remodelling in animal models and predicts incident AF in humans; however, a direct causal role for NOX2 in AF has not been demonstrated. Accordingly, we investigated whether myocardial NOX2 overexpression in mice (NOX2-Tg) is sufficient to generate a favourable substrate for AF and further assessed the effects of atorvastatin, an inhibitor of NOX2, on atrial superoxide production and AF susceptibility. METHODS AND RESULTS: NOX2-Tg mice showed a 2- to 2.5-fold higher atrial protein content of NOX2 compared with wild-type (WT) controls, which was associated with a significant (twofold) increase in NADPH-stimulated superoxide production (2-hydroxyethidium by HPLC) in left and right atrial tissue homogenates (P = 0.004 and P = 0.019, respectively). AF susceptibility assessed in vivo by transoesophageal atrial burst stimulation was modestly increased in NOX2-Tg compared with WT (probability of AF induction: 88% vs. 69%, respectively; P = 0.037), in the absence of significant alterations in AF duration, surface ECG parameters, and LV mass or function. Mechanistic studies did not support a role for NOX2 in promoting electrical or structural remodelling, as high-resolution optical mapping of atrial tissues showed no differences in action potential duration and conduction velocity between genotypes. In addition, we did not observe any genotype difference in markers of fibrosis and inflammation, including atrial collagen content and Col1a1, Il-1ß, Il-6, and Mcp-1 mRNA. Similarly, NOX2 overexpression did not have consistent effects on RyR2 Ca2+ leak nor did it affect PKA or CaMKII-mediated RyR2 phosphorylation. Finally, treatment with atorvastatin significantly inhibited atrial superoxide production in NOX2-Tg but had no effect on AF induction in either genotype. CONCLUSION: Together, these data indicate that while atrial NOX2 overexpression may contribute to atrial arrhythmogenesis, NOX2-derived superoxide production does not affect the electrical and structural properties of the atrial myocardium.

BH4 Increases nNOS Activity and Preserves Left Ventricular Function in Diabetes.

RATIONALE: In diabetic patients, heart failure with predominant left ventricular (LV) diastolic dysfunction is a common complication for which there is no effective treatment. Oxidation of the NOS (nitric oxide synthase) cofactor tetrahydrobiopterin (BH4) and dysfunctional NOS activity have been implicated in the pathogenesis of the diabetic vascular and cardiomyopathic phenotype. OBJECTIVE: Using mice models and human myocardial samples, we evaluated whether and by which mechanism increasing myocardial BH4 availability prevented or reversed LV dysfunction induced by diabetes. METHODS AND RESULTS: In contrast to the vascular endothelium, BH4 levels, superoxide production, and NOS activity (by liquid chromatography) did not differ in the LV myocardium of diabetic mice or in atrial tissue from diabetic patients. Nevertheless, the impairment in both cardiomyocyte relaxation and [Ca2+]i (intracellular calcium) decay and in vivo LV function (echocardiography and tissue Doppler) that developed in wild-type mice 12 weeks post-diabetes induction (streptozotocin, 42-45 mg/kg) was prevented in mGCH1-Tg (mice with elevated myocardial BH4 content secondary to trangenic overexpression of GTP-cyclohydrolase 1) and reversed in wild-type mice receiving oral BH4 supplementation from the 12th to the 18th week after diabetes induction. The protective effect of BH4 was abolished by CRISPR/Cas9-mediated knockout of nNOS (the neuronal NOS isoform) in mGCH1-Tg. In HEK (human embryonic kidney) cells, S-nitrosoglutathione led to a PKG (protein kinase G)-dependent increase in plasmalemmal density of the insulin-independent glucose transporter GLUT-1 (glucose transporter-1). In cardiomyocytes, mGCH1 overexpression induced a NO/sGC (soluble guanylate cyclase)/PKG-dependent increase in glucose uptake via GLUT-1, which was instrumental in preserving mitochondrial creatine kinase activity, oxygen consumption rate, LV energetics (by 31phosphorous magnetic resonance spectroscopy), and myocardial function. CONCLUSIONS: We uncovered a novel mechanism whereby myocardial BH4 prevents and reverses LV diastolic and systolic dysfunction associated with diabetes via an nNOS-mediated increase in insulin-independent myocardial glucose uptake and utilization. These findings highlight the potential of GCH1/BH4-based therapeutics in human diabetic cardiomyopathy. Graphic Abstract: A graphic abstract is available for this article.

Oxidation of Protein Kinase A Regulatory Subunit PKARIα Protects Against Myocardial Ischemia-Reperfusion Injury by Inhibiting Lysosomal-Triggered Calcium Release.

BACKGROUND: Kinase oxidation is a critical signaling mechanism through which changes in the intracellular redox state alter cardiac function. In the myocardium, PKARIα (type-1 protein kinase A) can be reversibly oxidized, forming interprotein disulfide bonds in the holoenzyme complex. However, the effect of PKARIα disulfide formation on downstream signaling in the heart, particularly under states of oxidative stress such as ischemia and reperfusion (I/R), remains unexplored. METHODS: Atrial tissue obtained from patients before and after cardiopulmonary bypass and reperfusion and left ventricular (LV) tissue from mice subjected to I/R or sham surgery were used to assess PKARIα disulfide formation by immunoblot. To determine the effect of disulfide formation on PKARIα catalytic activity and subcellular localization, live-cell fluorescence imaging and stimulated emission depletion super-resolution microscopy were performed in prkar1 knock-out mouse embryonic fibroblasts, neonatal myocytes, or adult LV myocytes isolated from "redox dead" (Cys17Ser) PKARIα knock-in mice and their wild-type littermates. Comparison of intracellular calcium dynamics between genotypes was assessed in fura2-loaded LV myocytes, whereas I/R-injury was assessed ex vivo. RESULTS: In both humans and mice, myocardial PKARIα disulfide formation was found to be significantly increased (2-fold in humans, P=0.023; 2.4-fold in mice, P<0.001) in response to I/R in vivo. In mouse LV cardiomyocytes, disulfide-containing PKARIα was not found to impact catalytic activity, but instead led to enhanced AKAP (A-kinase anchoring protein) binding with preferential localization of the holoenzyme to the lysosome. Redox-dependent regulation of lysosomal two-pore channels by PKARIα was sufficient to prevent global calcium release from the sarcoplasmic reticulum in LV myocytes, without affecting intrinsic ryanodine receptor leak or phosphorylation. Absence of I/R-induced PKARIα disulfide formation in "redox dead" knock-in mouse hearts resulted in larger infarcts (2-fold, P<0.001) and a concomitant reduction in LV contractile recovery (1.6-fold, P<0.001), which was prevented by administering the lysosomal two-pore channel inhibitor Ned-19 at the time of reperfusion. CONCLUSIONS: Disulfide modification targets PKARIα to the lysosome, where it acts as a gatekeeper for two-pore channel-mediated triggering of global calcium release. In the postischemic heart, this regulatory mechanism is critical for protection from extensive injury and offers a novel target for the design of cardioprotective therapeutics.

Modifications of Sarcoplasmic Reticulum Function Prevent Progression of Sarcomere-Linked Hypertrophic Cardiomyopathy Despite a Persistent Increase in Myofilament Calcium Response.

Hypertrophic cardiomyopathy (HCM) is a genetic disorder caused by mutations in different genes mainly encoding myofilament proteins and therefore called a "disease of the sarcomere." Despite the discovery of sarcomere protein mutations linked to HCM almost 30 years ago, the cellular mechanisms responsible for the development of this disease are not completely understood and likely vary among different mutations. Moreover, despite many efforts to develop effective treatments for HCM, these have largely been unsuccessful, and more studies are needed to better understand the cellular mechanisms of the disease. In experiments reported here, we investigated a mouse model expressing the mutant cTnT-R92Q, which is linked to HCM and induces an increase in myofilament Ca2+ sensitivity and diastolic dysfunction. We found that early correction of the diastolic dysfunction by phospholamban knockout (PLNKO) was able to prevent the development of the HCM phenotype in troponin T (TnT)-R92Q transgenic (TG) mice. Four groups of mice in FVB/N background were generated and used for the experiments: (1) non-transgenic (NTG)/PLN mice, which express wild-type TnT and normal level of PLN; (2) NTG/PLNKO mice, which express wild-type TnT and no PLN; (3) TG/PLN mice, which express TnT-R92Q and normal level of PLN; (4) TG/PLNKO mice, which express TnT-R92Q and no PLN. Cardiac function was determined using both standard echocardiographic parameters and speckle tracking strain measurements. We found that both atrial morphology and diastolic function were altered in TG/PLN mice but normal in TG/PLNKO mice. Histological analysis showed a disarray of myocytes and increased collagen deposition only in TG/PLN hearts. We also observed increased Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation only in TG/PLN hearts but not in TG/PLNKO hearts. The rescue of the HCM phenotype was not associated with differences in myofilament Ca2+ sensitivity between TG/PLN and TG/PLNKO mice. Moreover, compared to standard systolic echo parameters, such as ejection fraction (EF), speckle strain measurements provided a more sensitive approach to detect early systolic dysfunction in TG/PLN mice. In summary, our results indicate that targeting diastolic dysfunction through altering Ca2+ fluxes with no change in myofilament response to Ca2+ was able to prevent the development of the HCM phenotype and should be considered as a potential additional treatment for HCM patients.

Nitric oxide modulates cardiomyocyte pH control through a biphasic effect on sodium/hydrogen exchanger-1.

AIMS: When activated, Na+/H+ exchanger-1 (NHE1) produces some of the largest ionic fluxes in the heart. NHE1-dependent H+ extrusion and Na+ entry strongly modulate cardiac physiology through the direct effects of pH on proteins and by influencing intracellular Ca2+ handling. To attain an appropriate level of activation, cardiac NHE1 must respond to myocyte-derived cues. Among physiologically important cues is nitric oxide (NO), which regulates a myriad of cardiac functions, but its actions on NHE1 are unclear. METHODS AND RESULTS: NHE1 activity was measured using pH-sensitive cSNARF1 fluorescence after acid-loading adult ventricular myocytes by an ammonium prepulse solution manoeuvre. NO signalling was manipulated by knockout of its major constitutive synthase nNOS, adenoviral nNOS gene delivery, nNOS inhibition, and application of NO-donors. NHE1 flux was found to be activated by low [NO], but inhibited at high [NO]. These responses involved cGMP-dependent signalling, rather than S-nitros(yl)ation. Stronger cGMP signals, that can inhibit phosphodiesterase enzymes, allowed [cAMP] to rise, as demonstrated by a FRET-based sensor. Inferring from the actions of membrane-permeant analogues, cGMP was determined to activate NHE1, whereas cAMP was inhibitory, which explains the biphasic regulation by NO. Activation of NHE1-dependent Na+ influx by low [NO] also increased the frequency of spontaneous Ca2+ waves, whereas high [NO] suppressed these aberrant forms of Ca2+ signalling. CONCLUSIONS: Physiological levels of NO stimulation increase NHE1 activity, which boosts pH control during acid-disturbances and results in Na+-driven cellular Ca2+ loading. These responses are positively inotropic but also increase the likelihood of aberrant Ca2+ signals, and hence arrhythmia. Stronger NO signals inhibit NHE1, leading to a reversal of the aforementioned effects, ostensibly as a potential cardioprotective intervention to curtail NHE1 overdrive.

Oxidation resistance 1 regulates post-translational modifications of peroxiredoxin 2 in the cerebellum.

Protein aggregation, oxidative and nitrosative stress are etiological factors common to all major neurodegenerative disorders. Therefore, identifying proteins that function at the crossroads of these essential pathways may provide novel targets for therapy. Oxidation resistance 1 (Oxr1) is a protein proven to be neuroprotective against oxidative stress, although the molecular mechanisms involved remain unclear. Here, we demonstrate that Oxr1 interacts with the multifunctional protein, peroxiredoxin 2 (Prdx2), a potent antioxidant enzyme highly expressed in the brain that can also act as a molecular chaperone. Using a combination of in vitro assays and two animal models, we discovered that expression levels of Oxr1 regulate the degree of oligomerization of Prdx2 and also its post-translational modifications (PTMs), specifically suggesting that Oxr1 acts as a functional switch between the antioxidant and chaperone functions of Prdx2. Furthermore, we showed in the Oxr1 knockout mouse that Prdx2 is aberrantly modified by overoxidation and S-nitrosylation in the cerebellum at the presymptomatic stage; this in-turn affected the oligomerization of Prdx2, potentially impeding its normal functions and contributing to the specific cerebellar neurodegeneration in this mouse model.

Impaired cardiac contractile function in arginine:glycine amidinotransferase knockout mice devoid of creatine is rescued by homoarginine but not creatine.

AIMS: Creatine buffers cellular adenosine triphosphate (ATP) via the creatine kinase reaction. Creatine levels are reduced in heart failure, but their contribution to pathophysiology is unclear. Arginine:glycine amidinotransferase (AGAT) in the kidney catalyses both the first step in creatine biosynthesis as well as homoarginine (HA) synthesis. AGAT-/- mice fed a creatine-free diet have a whole body creatine-deficiency. We hypothesized that AGAT-/- mice would develop cardiac dysfunction and rescue by dietary creatine would imply causality. METHODS AND RESULTS: Withdrawal of dietary creatine in AGAT-/- mice provided an estimate of myocardial creatine efflux of ∼2.7%/day; however, in vivo cardiac function was maintained despite low levels of myocardial creatine. Using AGAT-/- mice naïve to dietary creatine we confirmed absence of phosphocreatine in the heart, but crucially, ATP levels were unchanged. Potential compensatory adaptations were absent, AMPK was not activated and respiration in isolated mitochondria was normal. AGAT-/- mice had rescuable changes in body water and organ weights suggesting a role for creatine as a compatible osmolyte. Creatine-naïve AGAT-/- mice had haemodynamic impairment with low LV systolic pressure and reduced inotropy, lusitropy, and contractile reserve. Creatine supplementation only corrected systolic pressure despite normalization of myocardial creatine. AGAT-/- mice had low plasma HA and supplementation completely rescued all other haemodynamic parameters. Contractile dysfunction in AGAT-/- was confirmed in Langendorff perfused hearts and in creatine-replete isolated cardiomyocytes, indicating that HA is necessary for normal cardiac function. CONCLUSIONS: Our findings argue against low myocardial creatine per se as a major contributor to cardiac dysfunction. Conversely, we show that HA deficiency can impair cardiac function, which may explain why low HA is an independent risk factor for multiple cardiovascular diseases.

Early sensitization of myofilaments to Ca2+ prevents genetically linked dilated cardiomyopathy in mice.

BACKGROUND: Dilated cardiomoypathies (DCM) are a heterogeneous group of inherited and acquired diseases characterized by decreased contractility and enlargement of cardiac chambers and a major cause of morbidity and mortality. Mice with Glu54Lys mutation in α-tropomyosin (Tm54) demonstrate typical DCM phenotype with reduced myofilament Ca2+ sensitivity. We tested the hypothesis that early sensitization of the myofilaments to Ca2+ in DCM can prevent the DCM phenotype. METHODS AND RESULTS: To sensitize Tm54 myofilaments, we used a genetic approach and crossbred Tm54 mice with mice expressing slow skeletal troponin I (ssTnI) that sensitizes myofilaments to Ca2+. Four groups of mice were used: non-transgenic (NTG), Tm54, ssTnI and Tm54/ssTnI (DTG). Systolic function was significantly reduced in the Tm54 mice compared to NTG, but restored in DTG mice. Tm54 mice also showed increased diastolic LV dimensions and HW/BW ratios, when compared to NTG, which were improved in the DTG group. ß-myosin heavy chain expression was increased in the Tm54 animals compared to NTG and was partially restored in DTG group. Analysis by 2D-DIGE indicated a significant decrease in two phosphorylated spots of cardiac troponin I (cTnI) in the DTG animals compared to NTG and Tm54. Analysis by 2D-DIGE also indicated no significant changes in troponin T, regulatory light chain, myosin binding protein C and tropomyosin phosphorylation. CONCLUSION: Our data indicate that decreased myofilament Ca2+ sensitivity is an essential element in the pathophysiology of thin filament linked DCM. Sensitization of myofilaments to Ca2+ in the early stage of DCM may be a useful therapeutic strategy in thin filament linked DCM.

Compromised redox homeostasis, altered nitroso-redox balance, and therapeutic possibilities in atrial fibrillation.

Although the initiation, development, and maintenance of atrial fibrillation (AF) have been linked to alterations in myocyte redox state, the field lacks a complete understanding of the impact these changes may have on cellular signalling, atrial electrophysiology, and disease progression. Recent studies demonstrate spatiotemporal changes in reactive oxygen species production shortly after the induction of AF in animal models with an uncoupling of nitric oxide synthase activity ensuing in the presence of long-standing persistent AF, ultimately leading to a major shift in nitroso-redox balance. However, it remains unclear which radical or non-radical species are primarily involved in the underlying mechanisms of AF or which proteins are targeted for redox modification. In most instances, only free radical oxygen species have been assessed; yet evidence from the redox signalling field suggests that non-radical species are more likely to regulate cellular processes. A wider appreciation for the distinction of these species and how both species may be involved in the development and maintenance of AF could impact treatment strategies. In this review, we summarize how redox second-messenger systems are regulated and discuss the recent evidence for alterations in redox regulation in the atrial myocardium in the presence of AF, while identifying some critical missing links. We also examine studies looking at antioxidants for the prevention and treatment of AF and propose alternative redox targets that may serve as superior therapeutic options for the treatment of AF.

Knockout of p21-activated kinase-1 attenuates exercise-induced cardiac remodelling through altered calcineurin signalling.

AIMS: Despite its known cardiovascular benefits, the intracellular signalling mechanisms underlying physiological cardiac growth remain poorly understood. Therefore, the purpose of this study was to investigate a novel role of p21-activated kinase-1 (Pak1) in the regulation of exercise-induced cardiac hypertrophy. METHODS AND RESULTS: Wild-type (WT) and Pak1 KO mice were subjected to 6 weeks of treadmill endurance exercise training (ex-training). Cardiac function was assessed via echocardiography, in situ haemodynamics, and the pCa-force relations in skinned fibre preparations at baseline and at the end of the training regimen. Post-translational modifications to the sarcomeric proteins and expression levels of calcium-regulating proteins were also assessed following ex-training. Heart weight/tibia length and echocardiography data revealed that there was marked hypertrophy following ex-training in the WT mice, which was not evident in the KO mice. Additionally, following ex-training, WT mice demonstrated an increase in cardiac contractility, myofilament calcium sensitivity, and phosphorylation of cardiac myosin-binding protein C, cardiac TnT, and tropomyosin compared with KO mice. With ex-training in WT mice, there were also increased protein levels of calcineurin and increased phosphorylation of phospholamban. CONCLUSIONS: Our data suggest that Pak1 is essential for adaptive physiological cardiac remodelling and support previous evidence that demonstrates Pak1 signalling is important for cardiac growth and survival.

Ceramide-mediated depression in cardiomyocyte contractility through PKC activation and modulation of myofilament protein phosphorylation.

Although ceramide accumulation in the heart is considered a major factor in promoting apoptosis and cardiac disorders, including heart failure, lipotoxicity and ischemia-reperfusion injury, little is known about ceramide's role in mediating changes in contractility. In the present study, we measured the functional consequences of acute exposure of isolated field-stimulated adult rat cardiomyocytes to C6-ceramide. Exogenous ceramide treatment depressed the peak amplitude and the maximal velocity of shortening without altering intracellular calcium levels or kinetics. The inactive ceramide analog C6-dihydroceramide had no effect on myocyte shortening or [Ca(2+)]i transients. Experiments testing a potential role for C6-ceramide-mediated effects on activation of protein kinase C (PKC) demonstrated evidence for signaling through the calcium-independent isoform, PKCε. We employed 2-dimensional electrophoresis and anti-phospho-peptide antibodies to test whether treatment of the cardiomyocytes with C6-ceramide altered myocyte shortening via PKC-dependent phosphorylation of myofilament proteins. Compared to controls, myocytes treated with ceramide exhibited increased phosphorylation of myosin binding protein-C (cMyBP-C), specifically at Ser273 and Ser302, and troponin I (cTnI) at sites apart from Ser23/24, which could be attenuated with PKC inhibition. We conclude that the altered myofilament response to calcium resulting from multiple sites of PKC-dependent phosphorylation contributes to contractile dysfunction that is associated with cardiac diseases in which elevations in ceramides are present.

Desensitization of myofilaments to Ca2+ as a therapeutic target for hypertrophic cardiomyopathy with mutations in thin filament proteins.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a common genetic disorder caused mainly by mutations in sarcomeric proteins and is characterized by maladaptive myocardial hypertrophy, diastolic heart failure, increased myofilament Ca(2+) sensitivity, and high susceptibility to sudden death. We tested the following hypothesis: correction of the increased myofilament sensitivity can delay or prevent the development of the HCM phenotype. METHODS AND RESULTS: We used an HCM mouse model with an E180G mutation in α-tropomyosin (Tm180) that demonstrates increased myofilament Ca(2+) sensitivity, severe hypertrophy, and diastolic dysfunction. To test our hypothesis, we reduced myofilament Ca(2+) sensitivity in Tm180 mice by generating a double transgenic mouse line. We crossed Tm180 mice with mice expressing a pseudophosphorylated cardiac troponin I (S23D and S24D; TnI-PP). TnI-PP mice demonstrated a reduced myofilament Ca(2+) sensitivity compared with wild-type mice. The development of pathological hypertrophy did not occur in mice expressing both Tm180 and TnI-PP. Left ventricle performance was improved in double transgenic compared with their Tm180 littermates, which express wild-type cardiac troponin I. Hearts of double transgenic mice demonstrated no changes in expression of phospholamban and sarcoplasmic reticulum Ca(2+) ATPase, increased levels of phospholamban and troponin T phosphorylation, and reduced phosphorylation of TnI compared with Tm180 mice. Moreover, expression of TnI-PP in Tm180 hearts inhibited modifications in the activity of extracellular signal-regulated kinase and zinc finger-containing transcription factor GATA in Tm180 hearts. CONCLUSIONS: Our data strongly indicate that reduction of myofilament sensitivity to Ca(2+) and associated correction of abnormal relaxation can delay or prevent development of HCM and should be considered as a therapeutic target for HCM.

Nitric oxide synthase regulation of cardiac excitation-contraction coupling in health and disease.

Significant advances in our understanding of the ability of nitric oxide synthases (NOS) to modulate cardiac function have provided key insights into the role NOS play in the regulation of excitation-contraction (EC) coupling in health and disease. Through both cGMP-dependent and cGMP-independent (e.g. S-nitrosylation) mechanisms, NOS have the ability to alter intracellular Ca(2+) handling and the myofilament response to Ca(2+), thereby impacting the systolic and diastolic performance of the myocardium. Findings from experiments using nitric oxide (NO) donors and NOS inhibition or gene deletion clearly implicate dysfunctional NOS as a critical contributor to many cardiovascular disease states. However, studies to date have only partially addressed NOS isoform-specific effects and, more importantly, how subcellular localization of NOS influences ion channels involved in myocardial EC coupling and excitability. In this review, we focus on the contribution of each NOS isoform to cardiac dysfunction and on the role of uncoupled NOS activity in common cardiac disease states, including heart failure, diabetic cardiomyopathy, ischemia/reperfusion injury and atrial fibrillation. We also review evidence that clearly indicates the importance of NO in cardioprotection. This article is part of a Special Issue entitled "Redox Signalling in the Cardiovascular System".