Nuclear localization of a novel calpain-2 mediated junctophilin-2 C-terminal cleavage peptide promotes cardiomyocyte remodeling.

Heart failure (HF) is a leading cause of morbidity and mortality worldwide. Patients with HF exhibit a loss of junctophilin-2 (JPH2), a structural protein critical in forming junctional membrane complexes in which excitation-contraction takes place. Several mechanisms have been proposed to mediate the loss of JPH2, one being cleavage by the calcium-dependent protease calpain. The downstream mechanisms underlying HF progression after JPH2 cleavage are presently poorly understood. In this study, we used Labcas to bioinformatically predict putative calpain cleavage sites on JPH2. We identified a cleavage site that produces a novel C-terminal JPH2 peptide (JPH2-CTP) using several domain-specific antibodies. Western blotting revealed elevated JPH2-CTP levels in hearts of patients and mice with HF, corresponding to increased levels of calpain-2. Moreover, immunocytochemistry demonstrated nuclear localization of JPH2-CTP within ventricular myocytes isolated from a murine model of pressure overload-induced HF as well as rat ventricular myocytes treated with isoproterenol. Nuclear localization of JPH2-CTP and cellular remodeling were abrogated by a genetic mutation of the nuclear localization sequence within JPH2-CTP. Taken together, our studies identified a novel C-terminal fragment of JPH2 (JPH2-CTP) generated by calpain-2 mediated cleavage which localizes within the cardiomyocyte nucleus during HF. Blocking nuclear localization of JPH2-CTP protects cardiomyocytes from isoproterenol-induced hypertrophy in vitro. Future in vivo studies of the nuclear role of JPH2-CTP may reveal a causal association with adverse remodeling during HF and establish CTP as a therapeutic target.

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.

The RNA-binding protein Rbm38 is dispensable during pressure overload-induced cardiac remodeling in mice.

The importance of tightly controlled alternative pre-mRNA splicing in the heart is emerging. The RNA binding protein Rbm24 has recently been identified as a pivotal cardiac splice factor, which governs sarcomerogenesis in the heart by controlling the expression of alternative protein isoforms. Rbm38, a homolog of Rbm24, has also been implicated in RNA processes such as RNA splicing, RNA stability and RNA translation, but its function in the heart is currently unknown. Here, we investigated the role of Rbm38 in the healthy and diseased adult mouse heart. In contrast to the heart- and skeletal muscle-enriched protein Rbm24, Rbm38 appears to be more broadly expressed. We generated somatic Rbm38 -/- mice and show that global loss of Rbm38 results in hematopoietic defects. Specifically, Rbm38 -/- mice were anemic and displayed enlarged spleens with extramedullary hematopoiesis, as has been shown earlier. The hearts of Rbm38 -/- mice were mildly hypertrophic, but cardiac function was not affected. Furthermore, Rbm38 deficiency did not affect cardiac remodeling (i.e. hypertrophy, LV dilation and fibrosis) or performance (i.e. fractional shortening) after pressure-overload induced by transverse aorta constriction. To further investigate molecular consequences of Rbm38 deficiency, we examined previously identified RNA stability, splicing, and translational targets of Rbm38. We found that stability targets p21 and HuR, splicing targets Mef2d and Fgfr2, and translation target p53 were not altered, suggesting that these Rbm38 targets are tissue-specific or that Rbm38 deficiency may be counteracted by a redundancy mechanism. In this regard, we found a trend towards increased Rbm24 protein expression in Rbm38 -/- hearts. Overall, we conclude that Rbm38 is critical in hematopoiesis, but does not play a critical role in the healthy and diseased heart.

A mutation in the glutamate-rich region of RNA-binding motif protein 20 causes dilated cardiomyopathy through missplicing of titin and impaired Frank-Starling mechanism.

AIM: Mutations in the RS-domain of RNA-binding motif protein 20 (RBM20) have recently been identified to segregate with aggressive forms of familial dilated cardiomyopathy (DCM). Loss of RBM20 in rats results in missplicing of the sarcomeric gene titin (TTN). The functional and physiological consequences of RBM20 mutations outside the mutational hotspot of RBM20 have not been explored to date. In this study, we investigated the pathomechanism of DCM caused by a novel RBM20 mutation in human cardiomyocytes. METHODS AND RESULTS: We identified a family with DCM carrying a mutation (RBM20(E913K/+)) in a glutamate-rich region of RBM20. Western blot analysis of endogenous RBM20 protein revealed strongly reduced protein levels in the heart of an RBM20(E913K/+ )carrier. RNA deep-sequencing demonstrated massive inclusion of exons coding for the spring region of titin in the RBM20(E913K/+ )carrier. Titin isoform analysis revealed a dramatic shift from the less compliant N2B towards the highly compliant N2BA isoforms in RBM20(E913K/+ )heart. Moreover, an increased sarcomere resting-length was observed in single cardiomyocytes and isometric force measurements revealed an attenuated Frank-Starling mechanism (FSM), which was rescued by protein kinase A treatment. CONCLUSION: A mutation outside the mutational hotspot of RBM20 results in haploinsufficiency of RBM20. This leads to disturbed alternative splicing of TTN, resulting in a dramatic shift to highly compliant titin isoforms and an impaired FSM. These effects may contribute to the early onset, and malignant course of DCM caused by RBM20 mutations. Altogether, our results demonstrate that heterozygous loss of RBM20 suffices to profoundly impair myocyte biomechanics by its disturbance of TTN splicing.

Long Non-Coding RNA Malat-1 Is Dispensable during Pressure Overload-Induced Cardiac Remodeling and Failure in Mice.

BACKGROUND: Long non-coding RNAs (lncRNAs) are a class of RNA molecules with diverse regulatory functions during embryonic development, normal life, and disease in higher organisms. However, research on the role of lncRNAs in cardiovascular diseases and in particular heart failure is still in its infancy. The exceptionally well conserved nuclear lncRNA Metastasis associated in lung adenocarcinoma transcript 1 (Malat-1) is a regulator of mRNA splicing and highly expressed in the heart. Malat-1 modulates hypoxia-induced vessel growth, activates ERK/MAPK signaling, and scavenges the anti-hypertrophic microRNA-133. We therefore hypothesized that Malat-1 may act as regulator of cardiac hypertrophy and failure during cardiac pressure overload induced by thoracic aortic constriction (TAC) in mice. RESULTS: Absence of Malat-1 did not affect cardiac hypertrophy upon pressure overload: Heart weight to tibia length ratio significantly increased in WT mice (sham: 5.78±0.55, TAC 9.79±1.82 g/mm; p<0.001) but to a similar extend also in Malat-1 knockout (KO) mice (sham: 6.21±1.12, TAC 8.91±1.74 g/mm; p<0.01) with no significant difference between genotypes. As expected, TAC significantly reduced left ventricular fractional shortening in WT (sham: 38.81±6.53%, TAC: 23.14±11.99%; p<0.01) but to a comparable degree also in KO mice (sham: 37.01±4.19%, TAC: 25.98±9.75%; p<0.05). Histological hallmarks of myocardial remodeling, such as cardiomyocyte hypertrophy, increased interstitial fibrosis, reduced capillary density, and immune cell infiltration, did not differ significantly between WT and KO mice after TAC. In line, the absence of Malat-1 did not significantly affect angiotensin II-induced cardiac hypertrophy, dysfunction, and overall remodeling. Above that, pressure overload by TAC significantly induced mRNA levels of the hypertrophy marker genes Nppa, Nppb and Acta1, to a similar extend in both genotypes. Alternative splicing of Ndrg2 after TAC was apparent in WT (isoform ratio; sham: 2.97±0.26, TAC 1.57±0.40; p<0.0001) and KO mice (sham: 3.64±0.37; TAC: 2.24±0.76; p<0.0001) and interestingly differed between genotypes both at baseline and after pressure overload (p<0.05 each). CONCLUSION: These findings confirm a role for the lncRNA Malat-1 in mRNA splicing. However, no critical role for Malat-1 was found in pressure overload-induced heart failure in mice, despite its reported role in vascularization, ERK/MAPK signaling, and regulation of miR-133.

Dyscholesterolemia Protects Against Ischemia-Induced Ventricular Arrhythmias.

BACKGROUND: Hypercholesterolemia protects against ventricular fibrillation in patients with myocardial infarction. We hypothesize that hypercholesterolemia protects against ischemia-induced reentrant arrhythmias because of altered ion channel function. METHODS AND RESULTS: ECGs were measured in low-density lipoprotein receptor knockout (LDLr(-/-)), apolipoprotein A1 knockout (ApoA1(-/-)), and wild-type (WT) mice. Action potentials, calcium handling, and ion currents were recorded in ventricular myocytes. Gene expression was determined by quantitative polymerase chain reaction and Western blot. In isolated perfused hearts, regional ischemia was induced and arrhythmia inducibility was tested. Serum low-density lipoprotein (LDL) cholesterol was higher in LDLr(-/-) mice than in WT mice (2.6 versus 0.4 mmol/L), and high-density lipoprotein cholesterol was significantly lower in ApoA1(-/-) mice than in WT mice (0.3 versus 1.8 mmol/L). LDLr(-/-) and ApoA1(-/-) myocytes contained more cholesterol than WT (34.4±2.8 and 36.5±2.4 versus 25.5±0.4 µmol/g protein). The major potassium currents were not different in LDLr(-/-) and ApoA1(-/-) compared with WT mice. The L-type calcium current (I(Ca)), however, was larger in LDLr(-/-) and ApoA1(-/-) than in WT (12.1±0.7 and 12.8±0.8 versus 9.4±1.1 pA/pF). Calcium transient amplitude and fractional sarcoplasmic reticulum calcium release were larger and action potential and QTc duration longer in LDLr(-/-) and ApoA1(-/-) than in WT mice (action potential duration at 90% of repolarization: 102±4 and 106±3 versus 84±3.1 ms; QTc: 50.9±1.3 and 52.8±0.8 versus 43.5±1.2 ms). During ischemia, ventricular tachycardia/ventricular fibrillation inducibility was larger in WT than in LDLr(-/-) and ApoA1(-/-) hearts. Expression of sodium channel and Ca-handling genes were not significantly different between groups. CONCLUSIONS: Dyscholesterolemia is associated with action potential prolongation because of increased I(Ca) and reduces occurrence of reentrant arrhythmias during ischemia.

Epac2 mediates cardiac ß1-adrenergic-dependent sarcoplasmic reticulum Ca2+ leak and arrhythmia.

BACKGROUND: ß-Adrenergic receptor (ß-AR) activation can provoke cardiac arrhythmias mediated by cAMP-dependent alterations of Ca(2+) signaling. However, cAMP can activate both protein kinase A and an exchange protein directly activated by cAMP (Epac), but their functional interaction is unclear. In heart, selective Epac activation can induce potentially arrhythmogenic sarcoplasmic reticulum (SR) Ca(2+) release that involves Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) effects on the ryanodine receptor (RyR). METHODS AND RESULTS: We tested whether physiological ß-AR activation causes Epac-mediated SR Ca(2+) leak and arrhythmias and whether it requires Epac1 versus Epac2, ß(1)-AR versus ß(2)-AR, and CaMKIIδ-dependent phosphorylation of RyR2-S2814. We used knockout (KO) mice for Epac1, Epac2, or both. All KOs exhibited unaltered basal cardiac function, Ca(2+) handling, and hypertrophy in response to pressure overload. However, SR Ca(2+) leak induced by the specific Epac activator 8-CPT in wild-type mice was abolished in Epac2-KO and double-KO mice but was unaltered in Epac1-KO mice. ß-AR-induced arrhythmias were also less inducible in Epac2-KO versus wild-type mice. ß-AR activation with protein kinase A inhibition mimicked 8-CPT effects on SR Ca(2+) leak and was prevented by blockade of ß(1)-AR but not ß(2)-AR. CaMKII inhibition (KN93) and genetic ablation of either CaMKIIδ or CaMKII phosphorylation on RyR2-S2814 prevented 8-CPT-induced SR Ca(2+) leak. CONCLUSIONS: ß(1)-AR activates Epac2 to induce SR Ca(2+) leak via CaMKIIδ-dependent phosphorylation of RyR2-S2814. This pathway contributes to ß-AR-induced arrhythmias and reduced cardiac function.

Role of RyR2 phosphorylation at S2814 during heart failure progression.

RATIONALE: Increased activity of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is thought to promote heart failure (HF) progression. However, the importance of CaMKII phosphorylation of ryanodine receptors (RyR2) in HF development and associated diastolic sarcoplasmic reticulum Ca(2+) leak is unclear. OBJECTIVE: Determine the role of CaMKII phosphorylation of RyR2 in patients and mice with nonischemic and ischemic forms of HF. METHODS AND RESULTS: Phosphorylation of the primary CaMKII site S2814 on RyR2 was increased in patients with nonischemic, but not with ischemic, HF. Knock-in mice with an inactivated S2814 phosphorylation site were relatively protected from HF development after transverse aortic constriction compared with wild-type littermates. After transverse aortic constriction, S2814A mice did not exhibit pulmonary congestion and had reduced levels of atrial natriuretic factor. Cardiomyocytes from S2814A mice exhibited significantly lower sarcoplasmic reticulum Ca(2+) leak and improved sarcoplasmic reticulum Ca(2+) loading compared with wild-type mice after transverse aortic constriction. Interestingly, these protective effects on cardiac contractility were not observed in S2814A mice after experimental myocardial infarction. CONCLUSIONS: Our results suggest that increased CaMKII phosphorylation of RyR2 plays a role in the development of pathological sarcoplasmic reticulum Ca(2+) leak and HF development in nonischemic forms of HF such as transverse aortic constriction in mice.

Disrupted junctional membrane complexes and hyperactive ryanodine receptors after acute junctophilin knockdown in mice.

BACKGROUND: Excitation-contraction coupling in striated muscle requires proper communication of plasmalemmal voltage-activated Ca2+ channels and Ca2+ release channels on sarcoplasmic reticulum within junctional membrane complexes. Although previous studies revealed a loss of junctional membrane complexes and embryonic lethality in germ-line junctophilin-2 (JPH2) knockout mice, it has remained unclear whether JPH2 plays an essential role in junctional membrane complex formation and the Ca(2+)-induced Ca(2+) release process in the heart. Our recent work demonstrated loss-of-function mutations in JPH2 in patients with hypertrophic cardiomyopathy. METHODS AND RESULTS: To elucidate the role of JPH2 in the heart, we developed a novel approach to conditionally reduce JPH2 protein levels using RNA interference. Cardiac-specific JPH2 knockdown resulted in impaired cardiac contractility, which caused heart failure and increased mortality. JPH2 deficiency resulted in loss of excitation-contraction coupling gain, precipitated by a reduction in the number of junctional membrane complexes and increased variability in the plasmalemma-sarcoplasmic reticulum distance. CONCLUSIONS: Loss of JPH2 had profound effects on Ca2+ release channel inactivation, suggesting a novel functional role for JPH2 in regulating intracellular Ca2+ release channels in cardiac myocytes. Thus, our novel approach of cardiac-specific short hairpin RNA-mediated knockdown of junctophilin-2 has uncovered a critical role for junctophilin in intracellular Ca2+ release in the heart.

Junctophilin-2 expression silencing causes cardiocyte hypertrophy and abnormal intracellular calcium-handling.

BACKGROUND: Junctophilin-2 (JPH2), a protein expressed in the junctional membrane complex, is necessary for proper intracellular calcium (Ca(2+)) signaling in cardiac myocytes. Downregulation of JPH2 expression in a model of cardiac hypertrophy was recently associated with defective coupling between plasmalemmal L-type Ca(2+) channels and sarcoplasmic reticular ryanodine receptors. However, it remains unclear whether JPH2 expression is altered in patients with hypertrophic cardiomyopathy (HCM). In addition, the effects of downregulation of JPH2 expression on intracellular Ca(2+) handling are presently poorly understood. We sought to determine whether loss of JPH2 expression is noted among patients with HCM and whether expression silencing might perturb Ca(2+) handling in a prohypertrophic manner. METHODS AND RESULTS: JPH2 expression was reduced in flash-frozen human cardiac tissue procured from patients with HCM compared with ostensibly healthy traumatic death victims. Partial silencing of JPH2 expression in HL-1 cells by a small interfering RNA probe targeted to murine JPH2 mRNA (shJPH2) resulted in myocyte hypertrophy and increased expression of known markers of cardiac hypertrophy. Whereas expression levels of major Ca(2+)-handling proteins were unchanged, shJPH2 cells demonstrated depressed maximal Ca(2+) transient amplitudes that were insensitive to L-type Ca(2+) channel activation with JPH2 knockdown. Further, reduced caffeine-triggered sarcoplasmic reticulum store Ca(2+) levels were observed with potentially increased total Ca(2+) stores. Spontaneous Ca(2+) oscillations were elicited at a higher extracellular [Ca(2+)] and with decreased frequency in JPH2 knockdown cells. CONCLUSIONS: Our results show that JPH2 levels are reduced in patients with HCM. Reduced JPH2 expression results in reduced excitation-contraction coupling gain as well as altered Ca(2+) homeostasis, which may be associated with prohypertrophic remodeling.

Ryanodine receptor phosphorylation by calcium/calmodulin-dependent protein kinase II promotes life-threatening ventricular arrhythmias in mice with heart failure.

BACKGROUND: approximately half of patients with heart failure die suddenly as a result of ventricular arrhythmias. Although abnormal Ca(2+) release from the sarcoplasmic reticulum through ryanodine receptors (RyR2) has been linked to arrhythmogenesis, the molecular mechanisms triggering release of arrhythmogenic Ca(2+) remain unknown. We tested the hypothesis that increased RyR2 phosphorylation by Ca(2+)/calmodulin-dependent protein kinase II is both necessary and sufficient to promote lethal ventricular arrhythmias. METHODS AND RESULTS: mice in which the S2814 Ca(2+)/calmodulin-dependent protein kinase II site on RyR2 is constitutively activated (S2814D) develop pathological sarcoplasmic reticulum Ca(2+) release events, resulting in reduced sarcoplasmic reticulum Ca(2+) load on confocal microscopy. These Ca(2+) release events are associated with increased RyR2 open probability in lipid bilayer preparations. At baseline, young S2814D mice have structurally and functionally normal hearts without arrhythmias; however, they develop sustained ventricular tachycardia and sudden cardiac death on catecholaminergic provocation by caffeine/epinephrine or programmed electric stimulation. Young S2814D mice have a significant predisposition to sudden arrhythmogenic death after transverse aortic constriction surgery. Finally, genetic ablation of the Ca(2+)/calmodulin-dependent protein kinase II site on RyR2 (S2814A) protects mutant mice from pacing-induced arrhythmias versus wild-type mice after transverse aortic constriction surgery. CONCLUSIONS: our results suggest that Ca(2+)/calmodulin-dependent protein kinase II phosphorylation of RyR2 Ca(2+) release channels at S2814 plays an important role in arrhythmogenesis and sudden cardiac death in mice with heart failure.

Genetic inhibition of PKA phosphorylation of RyR2 prevents dystrophic cardiomyopathy.

Aberrant intracellular Ca(2+) regulation is believed to contribute to the development of cardiomyopathy in Duchenne muscular dystrophy. Here, we tested whether inhibition of protein kinase A (PKA) phosphorylation of ryanodine receptor type 2 (RyR2) prevents dystrophic cardiomyopathy by reducing SR Ca(2+) leak in the mdx mouse model of Duchenne muscular dystrophy. mdx mice were crossed with RyR2-S2808A mice, in which PKA phosphorylation site S2808 on RyR2 is inactivated by alanine substitution. Compared with mdx mice that developed age-dependent heart failure, mdx-S2808A mice exhibited improved fractional shortening and reduced cardiac dilation. Whereas application of isoproterenol severely depressed cardiac contractility and caused 95% mortality in mdx mice, contractility was preserved with only 19% mortality in mdx-S2808A mice. SR Ca(2+) leak was greater in ventricular myocytes from mdx than mdx-S2808A mice. Myocytes from mdx mice had a higher incidence of isoproterenol-induced diastolic Ca(2+) release events than myocytes from mdx-S2808A mice. Thus, inhibition of PKA phosphorylation of RyR2 reduced SR Ca(2+) leak and attenuated cardiomyopathy in mdx mice, suggesting that enhanced PKA phosphorylation of RyR2 at S2808 contributes to abnormal Ca(2+) homeostasis associated with dystrophic cardiomyopathy.

Transverse aortic constriction in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart failure. TAC initially leads to compensated hypertrophy of the heart, which often is associated with a temporary enhancement of cardiac contractility. Over time, however, the response to the chronic hemodynamic overload becomes maladaptive, resulting in cardiac dilatation and heart failure. The murine TAC model was first validated by Rockman et al., and has since been extensively used as a valuable tool to mimic human cardiovascular diseases and elucidate fundamental signaling processes involved in the cardiac hypertrophic response and heart failure development. When compared to other experimental models of heart failure, such as complete occlusion of the left anterior descending (LAD) coronary artery, TAC provides a more reproducible model of cardiac hypertrophy and a more gradual time course in the development of heart failure. Here, we describe a step-by-step procedure to perform surgical TAC in mice. To determine the level of pressure overload produced by the aortic ligation, a high frequency Doppler probe is used to measure the ratio between blood flow velocities in the right and left carotid arteries. With surgical survival rates of 80-90%, transverse aortic banding is an effective technique of inducing left ventricular hypertrophy and heart failure in mice.

Accelerated development of pressure overload-induced cardiac hypertrophy and dysfunction in an RyR2-R176Q knockin mouse model.

In response to chronic hypertension, the heart compensates by hypertrophic growth, which frequently progresses to heart failure. Although intracellular calcium (Ca(2+)) has a central role in hypertrophic signaling pathways, the Ca(2+) source for activating these pathways remains elusive. We hypothesized that pathological sarcoplasmic reticulum Ca(2+) leak through defective cardiac intracellular Ca(2+) release channels/ryanodine receptors (RyR2) accelerates heart failure development by stimulating Ca(2+)-dependent hypertrophic signaling. Mice heterozygous for the gain-of-function mutation R176Q/+ in RyR2 and wild-type mice were subjected to transverse aortic constriction. Cardiac function was significantly lower, and cardiac dimensions were larger at 8 weeks after transverse aortic constriction in R176Q/+ compared with wild-type mice. R176Q/+ mice displayed an enhanced hypertrophic response compared with wild-type mice as assessed by heart weight:body weight ratios and cardiomyocyte cross-sectional areas after transverse aortic constriction. Quantitative PCR revealed increased transcriptional activation of cardiac stress genes in R176Q/+ mice after transverse aortic constriction. Moreover, pressure overload resulted in an increased sarcoplasmic reticulum Ca(2+) leak, associated with higher expression levels of the exon 4 splice form of regulator of calcineurin 1, and a decrease in nuclear factor of activated T-cells phosphorylation in R176Q/+ mice compared with wild-type mice. Taken together, our results suggest that RyR2-dependent sarcoplasmic reticulum Ca(2+) leak activates the prohypertrophic calcineurin/nuclear factor of activated T-cells pathway under conditions of pressure overload.

MEF2 transcriptional activity maintains mitochondrial adaptation in cardiac pressure overload.

AIMS: The transcription factor MEF2 is a downstream target for several hypertrophic signalling pathways in the heart, suggesting that MEF2 may act as a valuable therapeutic target in the treatment of heart failure. METHODS AND RESULTS: In this study, we investigated the potential benefits of overall MEF2 inhibition in a mouse model of chronic pressure overloading, by subjecting transgenic mice expressing a dominant negative form of MEF2 (DN-MEF2 Tg) in the heart, to transverse aortic constriction (TAC). Histological analysis revealed no major differences in cardiac remodelling between DN-MEF2 Tg and control mice after TAC. Surprisingly, echocardiographic analysis revealed that DN-MEF2 Tg mice had a decrease in cardiac function compared with control animals. Analysis of the mitochondrial respiratory chain showed that DN-MEF2 Tg mice displayed lower expression of NADH dehydrogenase subunit 6 (ND6), part of mitochondrial Complex I. The reduced expression of ND6 in DN-MEF2 Tg mice after pressure overload correlated with an increase in cell death secondary to overproduction of reactive oxygen species (ROS). CONCLUSION: Our data suggest that MEF2 transcriptional activity is required for mitochondrial function and its inhibition predisposes the heart to impaired mitochondrial function, overproduction of ROS, enhanced cell death, and cardiac dysfunction, following pressure overload.

Sudden infant death syndrome in mice with an inherited mutation in RyR2.

BACKGROUND: Mutations in the cardiac ryanodine receptor gene (RyR2) have been recently identified in victims of sudden infant death syndrome. The aim of this study was to determine whether a gain-of-function mutation in RyR2 increases the propensity to cardiac arrhythmias and sudden death in young mice. METHODS AND RESULTS: Incidence of sudden death was monitored prospectively in heterozygous knock-in mice with mutation R176Q in RyR2 (R176Q/+). Young R176Q/+ mice exhibited a higher incidence of sudden death compared with wild-type littermates. Optical mapping of membrane potentials and intracellular calcium in 1- to 7-day-old R176Q/+ and wild-type mice revealed an increased incidence of ventricular ectopy and spontaneous calcium releases in neonatal R176Q/+ mice. Surface ECGs in 3- to 10-day-old mice showed that R176Q/+ mice developed more ventricular arrhythmias after provocation with epinephrine and caffeine. Intracardiac pacing studies in 12- to 18-day-old mice revealed the presence of an arrhythmogenic substrate in R176Q/+ compared with wild-type mice. Reverse transcription-polymerase chain reaction and Western blotting showed that expression levels of other calcium handling proteins were unaltered, suggesting that calcium leak through mutant RyR2 underlies arrhythmogenesis and sudden death in young R176Q/+ mice. CONCLUSIONS: Our findings demonstrate that a gain-of-function mutation in RyR2 confers an increased risk of cardiac arrhythmias and sudden death in young mice and that young R176Q/+ mice may be used as a model for elucidating the complex interplay between genetic and environmental risk factors associated with sudden infant death syndrome.

Calmodulin kinase II-mediated sarcoplasmic reticulum Ca2+ leak promotes atrial fibrillation in mice.

A trial fibrillation (AF), the most common human cardiac arrhythmia, is associated with abnormal intracellular Ca2+ handling. Diastolic Ca2+ release from the sarcoplasmic reticulum via "leaky" ryanodine receptors (RyR2s) is hypothesized to contribute to arrhythmogenesis in AF, but the molecular mechanisms are incompletely understood. Here, we have shown that mice with a genetic gain-of-function defect in Ryr2 (which we termed Ryr2R176Q/+ mice) did not exhibit spontaneous AF but that rapid atrial pacing unmasked an increased vulnerability to AF in these mice compared with wild-type mice. Rapid atrial pacing resulted in increased Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of RyR2, while both pharmacologic and genetic inhibition of CaMKII prevented AF inducibility in Ryr2R176Q/+ mice. This result suggests that AF requires both an arrhythmogenic substrate (e.g., RyR2 mutation) and enhanced CaMKII activity. Increased CaMKII phosphorylation of RyR2 was observed in atrial biopsies from mice with atrial enlargement and spontaneous AF, goats with lone AF, and patients with chronic AF. Genetic inhibition of CaMKII phosphorylation of RyR2 in Ryr2S2814A knockin mice reduced AF inducibility in a vagotonic AF model. Together, these findings suggest that increased RyR2-dependent Ca2+ leakage due to enhanced CaMKII activity is an important downstream effect of CaMKII in individuals susceptible to AF induction.

Molecular evolution of the junctophilin gene family.

Junctophilins (JPHs) are members of a junctional membrane complex protein family important for the physical approximation of plasmalemmal and sarcoplasmic/endoplasmic reticulum membranes. As such, JPHs facilitate signal transduction in excitable cells between plasmalemmal voltage-gated calcium channels and intracellular calcium release channels. To determine the molecular evolution of the JPH gene family, we performed a phylogenetic analysis of over 60 JPH genes from over 40 species and compared conservation across species and different isoforms. We found that JPHs are evolutionary highly conserved, in particular the membrane occupation and recognition nexus motifs found in all species. Our data suggest that an ancestral form of JPH arose at the latest in a common metazoan ancestor and that in vertebrates four isoforms arose, probably following two rounds of whole genome duplications. By combining multiple prediction techniques with sequence alignments, we also postulate the presence of new important functional regions and candidate sites for posttranslational modifications. The increasing number of available sequences yields significant insight into the molecular evolution of JPHs. Our analysis is consistent with the emerging concept that JPHs serve dual important functions in excitable cells: structural assembly of junctional membrane complexes and regulation of intracellular calcium signaling pathways.