Background Human mutations in the X‐linked lysosome‐associated membrane protein‐2 (LAMP2) gene can cause a multisystem Danon disease or a primary cardiomyopathy characterized by massive hypertrophy, conduction system abnormalities, and malignant ventricular arrhythmias. We introduced an in‐frame LAMP2 gene exon 6 deletion mutation (denoted L2Δ6) causing human cardiomyopathy, into mouse LAMP2 gene, to elucidate its consequences on cardiomyocyte biology. This mutation results in in‐frame deletion of 41 amino acids, compatible with presence of some defective LAMP2 protein. Methods and Results Left ventricular tissues from L2Δ6 and wild‐type mice had equivalent amounts of LAMP2 RNA, but a significantly lower level of LAMP2 protein. By 20 weeks of age male mutant mice developed left ventricular hypertrophy which was followed by left ventricular dilatation and reduced systolic function. Cardiac electrophysiology and isolated cardiomyocyte studies demonstrated ventricular arrhythmia, conduction disturbances, abnormal calcium transients and increased sensitivity to catecholamines. Myocardial fibrosis was strikingly increased in 40‐week‐old L2Δ6 mice, recapitulating findings of human LAMP2 cardiomyopathy. Immunofluorescence and transmission electron microscopy identified mislocalization of lysosomes and accumulation of autophagosomes between sarcomeres, causing profound morphological changes disrupting the cellular ultrastructure. Transcription profile and protein expression analyses of L2Δ6 hearts showed significantly increased expression of genes encoding activators and protein components of autophagy, hypertrophy, and apoptosis. Conclusions We suggest that impaired autophagy results in cardiac hypertrophy and profound transcriptional reactions that impacted metabolism, calcium homeostasis, and cell survival. These responses define the molecular pathways that underlie the pathology and aberrant electrophysiology in cardiomyopathy of Danon disease.

Background: The transcription factor NKX2-5 is crucial for heart development and mutations in this gene have been implicated in diverse congenital heart diseases (CHD) and conduction defects (CD) in mouse models and humans. Whether NKX2-5 mutations have a role in adult-onset heart disease is unknown. Methods and Results: Mutation screening was performed in 220 probands with adult-onset dilated cardiomypathy (DCM). Six NKX2-5 coding sequence variants were identified, including 3 non-synonymous variants. A novel heterozygous mutation, I184M, located within the NKX2-5 homeodomain (HD), was identified in one family. A subset of family members had CHD, but there was an unexpectedly high prevalence of DCM. Functional analysis of I184M in vitro demonstrated a striking increase in protein expression when transfected into COS-7 cells or HL-1 cardiomyocytes, due to reduced degradation by the ubiquitinproteasome system (UPS). In functional assays, DNA binding activity of I184M was reduced, resulting in impaired activation of target genes, despite increased expression levels of mutant protein. Conclusions: Certain NKX2-5 HD mutations show abnormal protein degradation via the UPS and partially impaired transcriptional activity. We propose that this class of mutation can impair heart development and mature heart function, and contribute to NKX2-5-related cardiomyopathies with graded severity.

BACKGROUND:The transcription factor NKX2-5 is crucial for heart development, and mutations in this gene have been implicated in diverse congenital heart diseases and conduction defects in mouse models and humans. Whether NKX2-5 mutations have a role in adult-onset heart disease is unknown. METHODS AND RESULTS:Mutation screening was performed in 220 probands with adult-onset dilated cardiomyopathy. Six NKX2-5 coding sequence variants were identified, including 3 nonsynonymous variants. A novel heterozygous mutation, I184M, located within the NKX2-5 homeodomain, was identified in 1 family. A subset of family members had congenital heart disease, but there was an unexpectedly high prevalence of dilated cardiomyopathy. Functional analysis of I184M in vitro demonstrated a striking increase in protein expression when transfected into COS-7 cells or HL-1 cardiomyocytes because of reduced degradation by the Ubiquitin-proteasome system. In functional assays, DNA-binding activity of I184M was reduced, resulting in impaired activation of target genes despite increased expression levels of mutant protein. CONCLUSIONS:Certain NKX2-5 homeodomain mutations show abnormal protein degradation via the Ubiquitin-proteasome system and partially impaired transcriptional activity. We propose that this class of mutation can impair heart development and mature heart function and contribute to NKX2-5-related cardiomyopathies with graded severity.

BACKGROUNDDilated cardiomyopathy and hypertrophic cardiomyopathy arise from mutations in many genes. TTN, the gene encoding the sarcomere protein titin, has been insufficiently analyzed for cardiomyopathy mutations because of its enormous size.METHODSWe analyzed TTN in 312 subjects with dilated cardiomyopathy, 231 subjects with hypertrophic cardiomyopathy, and 249 controls by using next-generation or dideoxy sequencing. We evaluated deleterious variants for cosegregation in families and assessed clinical characteristics.RESULTSWe identified 72 unique mutations (25 nonsense, 23 frameshift, 23 splicing, and 1 large tandem insertion) that altered full-length titin. Among subjects studied by means of next-generation sequencing, the frequency of TTN mutations was significantly higher among subjects with dilated cardiomyopathy (54 of 203 [27%]) than among subjects with hypertrophic cardiomyopathy (3 of 231 [1%], P=3×10(-16)) or controls (7 of 249 [3%], P=9×10(-14)). TTN mutations cosegregated with dilated cardiomyopathy in families (combined lod score, 11.1) with high (>95%) observed penetrance after the age of 40 years. Mutations associated with dilated cardiomyopathy were overrepresented in the titin A-band but were absent from the Z-disk and M-band regions of titin (P≤0.01 for all comparisons). Overall, the rates of cardiac outcomes were similar in subjects with and those without TTN mutations, but adverse events occurred earlier in male mutation carriers than in female carriers (P=4×10(-5)).CONCLUSIONSTTN truncating mutations are a common cause of dilated cardiomyopathy, occurring in approximately 25% of familial cases of idiopathic dilated cardiomyopathy and in 18% of sporadic cases. Incorporation of sequencing approaches that detect TTN truncations into genetic testing for dilated cardiomyopathy should substantially increase test sensitivity, thereby allowing earlier diagnosis and therapeutic intervention for many patients with dilated cardiomyopathy. Defining the functional effects of TTN truncating mutations should improve our understanding of the pathophysiology of dilated cardiomyopathy. (Funded by the Howard Hughes Medical Institute and others.).

Method and results: CPVT mouse models that lack CASQ2 were treated with Ca2+-channel inhibitors, beta-adrenergic inhibitors, or Mg2+. Treatment effects on ventricular arrhythmia, sarcoplasmic reticulum (SR) protein expression and Ca2+ transients of isolated myocytes were assessed. Each study agent reduced the frequency of stress-induced ventricular arrhythmia in mutant mice. The Ca2+ channel blocker verapamil was most efficacious and completely prevented arrhythmia in 85% of mice. Verapamil significantly increased the SR Ca2+ content in mutant myocytes, diminished diastolic Ca2+ overload, increased systolic Ca2+ amplitude, and prevented Ca2+ oscillations in stressed mutant myocytes.Conclusions: Ca2+ channel inhibition by verapamil rectified abnormal calcium handling in CPVT myocytes and prevented ventricular arrhythmias. Verapamil-induced partial normalization of SR Ca2+ content in mutant myocytes implicates CASQ2 as modulator of RyR2 activity, rather than or in addition to, Ca2+ buffer protein. Agents such as verapamil that attenuate cardiomyocyte calcium overload are appropriate for assessing clinical efficacy in human CPVT. (J Cardiovasc Electrophysiol, Vol. 22, pp. 316-324, March 2011).

Unknown molecular responses to sarcomere protein gene mutations account for pathologic remodeling in hypertrophic cardiomyopathy (HCM), producing myocyte growth and increased cardiac fibrosis. To determine if hypertrophic signals activated myocyte enhancer factor-2 (Mef2), we studied mice carrying the HCM mutation, myosin heavy-chain Arg403Gln, (MHC 403/+ ) and an Mef2-dependent β-galactosidase reporter transgene. In young, prehypertrophic MHC 403/+ mice the reporter was not activated. In hypertrophic hearts, activation of the Mef2-dependent reporter was remarkably heterogeneous and was observed consistently in myocytes that bordered fibrotic foci with necrotic cells, MHC 403/+ myocytes with Mef2-dependent reporter activation reexpressed the fetal myosin isoform (βMHC), a molecular marker of hypertrophy, although MHC 403/+ myocytes with or without βMHC expression were comparably enlarged over WT myocytes. To consider Mef2 roles in severe HCM, we studied homozygous MHC 403/403 mice, which have accelerated remodeling, widespread myocyte necrosis, and neonatal lethality. Levels of phosphorylated class II histone deacetylases that activate Mef2 were substantially increased in MHC 403/403 hearts, but Mef2-dependent reporter activation was patchy. Sequential analyses showed myocytes increased Mef2-dependent reporter activity before death. Our data dissociate myocyte hypertrophy, a consistent response in HCM, from heterogeneous Mef2 activation and reexpression of a fetal gene program. The temporal and spatial relationship of Mef2-dependent gene activation with myocyte necrosis and fibrosis in MHC 403/+ and MHC 403/403 hearts defines Mef2 activation as a molecular signature of stressed HCM myocytes that are poised to die.

Unexplained cardiac hypertrophy, the diagnostic criterion for hypertrophic cardiomyopathy (HCM), occurs in 1 in 500 adults. Insights into the genetic cause and molecular pathophysiology of HCM are reshaping clinical paradigms for diagnosis and treatment of this common myocardial disorder. Human genetic studies have established that dominant mutations in the proteins that make up the contractile apparatus (the sarcomere) cause HCM. With the current availability of clinical gene-based diagnostics, pathogenic mutations in affected patients can be defined, which can suggest a clinical course and allow definitive preclinical identification of family members at risk for HCM. Genetic discoveries have also fostered mechanistic investigations in model organisms that are engineered to carry human HCM mutations. Novel therapeutic targets have emerged from these fundamental studies and are currently under clinical assessment in humans. The combination of contemporary gene-based diagnosis with new strategies to attenuate disease development and progression is changing the natural history of lifelong cardiac symptoms, arrhythmias, and heart failure from HCM.

Mutations in sarcomere protein genes can cause hypertrophic cardiomyopathy (HCM), a disorder characterized by myocyte enlargement, fibrosis, and impaired ventricular relaxation. Here, we demonstrate that sarcomere protein gene mutations activate proliferative and profibrotic signals in non-myocyte cells to produce pathologic remodeling in HCM. Gene expression analyses of non-myocyte cells isolated from HCM mouse hearts showed increased levels of RNAs encoding cell-cycle proteins, Tgf-beta, periostin, and other profibrotic proteins. Markedly increased BrdU labeling, Ki67 antigen expression, and periostin immunohistochemistry in the fibrotic regions of HCM hearts confirmed the transcriptional profiling data. Genetic ablation of periostin in HCM mice reduced but did not extinguish non-myocyte proliferation and fibrosis. In contrast, administration of Tgf-beta-neutralizing antibodies abrogated non-myocyte proliferation and fibrosis. Chronic administration of the angiotensin II type I receptor antagonist losartan to mutation-positive, hypertrophy-negative (prehypertrophic) mice prevented the emergence of hypertrophy, non-myocyte proliferation, and fibrosis. Losartan treatment did not reverse pathologic remodeling of established HCM but did reduce non-myocyte proliferation. These data define non-myocyte activation of Tgf-beta signaling as a pivotal mechanism for increased fibrosis in HCM and a potentially important factor contributing to diastolic dysfunction and heart failure. Preemptive pharmacologic inhibition of Tgf-beta signals warrants study in human patients with sarcomere gene mutations.

Hypertrophy cardiomyopathy (HCM) is an autosomal dominant disorder characterized by increased heart mass that occurs without a defined stimulus (such as hypertension or valvular disease). It is commonly recognized through the widespread use of non-invasive imaging. Epidemiological studies indicate that 1 of 500 individuals has unexplained cardiac hypertrophy, an observation that predicts a considerable role for genetics in this enigmatic disorder. Indeed, to date, more than 500 mutations had been identified in more than 12 genes encoding components of the thick and thin filament of the sarcomere and other myofilament-related proteins. Intensive studies of HCM continue to take our understandings about this fascinating disease in new directions. Mechanistic analyses have provided insights into how mutational alterations in these structural proteins may trigger the hypertrophic remodeling processes and other associated clinical features of HCM. Based on these studies, investigations have been initiated to assess whether early pharmacological interventions could prevent or attenuate the development of the disease and its clinical sequelae. By combining pathophysiology with knowledge of genetic cause and molecular responses, HCM has begun to exemplify opportunities for predictive and personalized medicine. With the emergence of newer technologies that enable high-throughput sequencing of DNA, it is timely to review clinical manifestations and genetic causes of this unique disease, and how intertwining these insights can improve contemporary diagnosis and management of HCM and other genetic forms of cardiac hypertrophy.

Catecholaminergic polymorphic ventricular tachycardia (CPVT), a familial arrhythmia syndrome caused by ryanodine receptor (RyR2) or calsequestrin-2 (CASQ2) gene mutations, is characterized by stres...

Background— Pheochromocytomas are rare tumors derived from the chromaffin cells of the adrenal medulla. Although these tumors have long been postulated to induce hypertension and cardiomyopathy through the hypersecretion of catecholamines, catecholamines alone may not fully explain the profound myocardial remodeling induced by these tumors. We sought to determine whether changes in myocardial function in pheochromocytoma-induced cardiomyopathy result solely from catecholamines secretion or from multiple pheochromocytoma-derived factors. Methods and Results— Isolated cardiomyocytes incubated with pheochromocytoma-conditioned growth media contracted at a higher frequency than cardiomyocytes incubated with norepinephrine (NE) only. Sprague-Dawley rats and black-6 mice were implanted with agarose-encapsulated pheochromocytoma (PC12) cells, dihydroxyphenylalanine decarboxylase knock-out PC12 cells deficient in NE (PC12-KO), or NE-secreting pumps. PC12 cell implantation increased left ventricular dilation by 35�6% and 9.6�1.4% and reduced left ventricular fractional shortening by 20�3% and 28�4% in rats and mice compared with animals dosed only with NE, respectively. Elimination of NE secretion in PC12-KO cells induced neither cardiac dilation (3.9%�1.8% increase versus control) nor changes in (1.9%�0.4% reduction) fractional shortening compared to controls. Conclusions— Pheochromocytomas induce a greater degree of cardiomyopathy than equivalent doses of NE, suggesting pheochromocytoma-induced cardiomyopathy is not solely mediated by NE, rather pheochromocytoma secretory factors in combination with catecholamines act synergistically to induce greater cardiac damage than catecholamines alone.

Background- The molecular triggers activated by a sarcomere gene mutation that remodel the heart and produce histopathologic manifestations of hypertrophic cardiomyopathy (HCM) are incompletely known. Myocyte enhancer factor 2 (MEF2) has emerged as a critical regulator of gene expression in the heart. Objective- To examine whether MEF2 is essential for HCM pathogenesis, we assessed in vivo MEF2 expression versus myocyte hypertrophy, disarray and interstitial fibrosis, the prototypic histopathology of HCM. Methods and Results- Wild-type or heterozygous mice harboring an Arg403Gln missense mutation in the alpha-myosin heavy chain gene (MHC403/+ mice) were crossed with transgenic mice harboring a MEF2-dependent reporter gene. Mouse hearts at 30 weeks of the age were evaluated by beta-galactosidase staining to detect transcriptional activity of MEF2 in vivo. Activity of MEF2 was heterogeneously increased in the left ventricle of MHC403/+ mouse hearts compared with wild-type mouse hearts. Gomori’s trichrome staining for collagen revealed that regional activation of MEF2 uniquely colocalized with myocardial scarring in MHC403/+ mouse hearts whereas only baseline activity of MEF2 was detected in the area without myocardial scarring. von Kossa staining revealed myocardial necrosis near to MEF2 staining. To further explore whether MEF2 activity is involved in the pathogenesis of myocardial necrosis, mice carrying a homozygous Arg403Gln mutation (MHC403/403) and the MEF2-dependent reporter gene were analyzed. MEF2 was regionally activated in 3-days old MHC403/403 mouse hearts, in advance of the myocardial necrosis that occurs in homozygous mutant hearts. At 6-days, greater MEF2 activity was observed compared with 3-days old MHC403/403 hearts, and massive necrosis was present where MEF2 was activated. Conclusions- These findings provide the first evidence for regional activation of the MEF2-pathway is involved in evolution of myocardial fibrosis and necrosis in HCM. Because myocyte loss and myocardial scarring is implicated in the progression of ventricular dysfunction, these data indicate that strategies to prevent MEF2 activation may attenuate remodeling and heart failure development.

To investigate the mechanisms by which mutations in the human transcriptional co-activator EYA4 gene cause sensorineural hearing loss that can occur in association with dilated cardiomyopathy, we studied eya4 expression during zebrafish development and characterized eya4 deficiency. eya4 morphant fish embryos had reduced numbers of hair cells in the otic vesicle and lateral line neuromasts with impaired sensory responses. Analyses of candidate genes that are known to be expressed in a temporal and spatial pattern comparable to eya4focused our analyses on atp1b2b, which encodes the β2b subunit of the zebrafish Na+/K+-ATPase. We demonstrate atp1b2b levels are reduced in eya4 morphant fish and that morpholino oligonucleotides targeting the atp1b2b gene recapitulated the eya4 deficiency phenotypes, including heart failure, decreased sensory hair cell numbers in the otic vesicle and neuromasts, and abnormal sensory responses. Furthermore, atp1b2b overexpression rescued these phenotypes in eya4 morphant fish. We conclude that eya4regulation of Na+/K+-ATPase is crucial for the development of mechanosensory cells and the maintenance of cardiac function in zebrafish.

1.1. BackgroundDanon disease is a poorly understood syndrome that consists of severe cardiomyopathy, skeletal muscle weakness and variable degree of learning disabilities [1]. Mutations in the gene encoding the lysosomal-associated membrane protein 2 (LAMP2) on chromosome X have been identified to cause Danon disease[2]. Although males are predominantly affected, unfortunate X-inactivation account for disease in some females. Recent genetic studies demonstrated that LAMP2 mutations account for profound, unexplained left ventricular hypertrophy (LVH) in children and young adults and can be detected in about 2-4% of children with unexplained LVH [3]. In addition to hypertrophy, these children may have electrophysiological abnormalities including life-threatening arrhythmias [3]. Gene-based diagnosis is essential to discriminate LAMP2 cardiomyopathy from hypertrophic cardiomyopathy so that clinical management and family counseling is appropriate.Although Danon syndrome was described more than two decades ago the mechanism of this type of cardiomyopathy is still obscure. Since muscle biopsies from affected subject demonstrated vacuoles positive for carbohydrate staining, this syndrome was considered as a subtype of glycogen storage disease [1], nevertheless none of the enzymes related to glycogen metabolism was found to be affected [4]. The discovery of mutations in the LAMP2 gene in Danon's patients led to further understanding of this syndrome. LAMP2 is a highly glycosylated protein that is an important constituent of the lysosomal membrane [5]. It is ubiquitously expressed but its function is unknown. A LAMP2 knock-out mouse model was found to have severe systemic phenotype with reduced weight and high rate of mortality up to 6 W [6]. It also exhibited excessive accumulation of autophagosomes in the liver, kidney, pancreas, heart and skeletal muscle [6].1.2. Methods and resultsTo better define how LAMP2 mutations cause cardiomyopathy we produced a mouse model that carries the human Del6 mutation (L26-) in the endogenous gene. About 20% of the L26-hemizygous male mice died during the first days after birth. The surviving L26-mice had significantly reduced weight up to 10th week but then they recovered, gain weight as their WT littermates and had near normal life span. L26-hemizygous mice developed cardiomyopathy at the age of 20 weeks with hypertrophy, dilatation, reduced contractile function and conduction system abnormalities typically seen in Danons' patients. Cardiac protein studies showed markedly reduced LAMP2 levels but no change in the levels of LAMP1. Glycogen assays showed only mild increased in the cardiac content of glycogen (1.5 fold). Histology studies demonstrated vacuoles accumulation, increased fibrosis but no signs of disarray. Electron microscopy of the ventricular heart muscle cells demonstrated the presence of large autophagic vacuoles filled with amorphic material and cytoplasmic degradation products similar to findings described by Tanaka et al [6].1.3. ConclusionWe concluded that LAMP2 cardiomyopathy is a result of partial LAMP2 deficiency within the lysosomal membrane which leads to accumulation of autophagic vacuoles and distraction of cardiomyocytes. Ongoing studies on this mouse model which recapitulates the human LAMP2 cardiomyopathy will provide further insight about disease mechanisms and may offer a model for development of rationale treatment strategies.

Mutations in the lamin A/C (LMNA) gene, which encodes nuclear membrane proteins, cause a variety of human conditions including dilated cardiomyopathy (DCM) with associated cardiac conduction system disease. To investigate mechanisms responsible for electrophysiologic and myocardial phenotypes caused by dominant human LMNA mutations, we performed longitudinal evaluations in heterozygous Lmna(+/-) mice. Despite one normal allele, Lmna(+/-) mice had 50% of normal cardiac lamin A/C levels and developed cardiac abnormalities. Conduction system function was normal in neonatal Lmna(+/-) mice but, by 4 weeks of age, atrioventricular (AV) nodal myocytes had abnormally shaped nuclei and active apoptosis. Telemetric and in vivo electrophysiologic studies in 10-week-old Lmna(+/-) mice showed AV conduction defects and both atrial and ventricular arrhythmias, analogous to those observed in humans with heterozygous LMNA mutations. Isolated myocytes from 12-month-old Lmna(+/-) mice exhibited impaired contractility. In vivo cardiac studies of aged Lmna(+/-) mice revealed DCM; in some mice this occurred without overt conduction system disease. However, neither histopathology nor serum CK levels indicated skeletal muscle pathology. These data demonstrate cardiac pathology due to heterozygous Lmna mutations reflecting a 50% reduction in lamin protein levels. Lamin haploinsufficiency caused early-onset programmed cell death of AV nodal myocytes and progressive electrophysiologic disease. While lamin haploinsufficiency was better tolerated by non-conducting myocytes, ultimately, these too succumbed to diminished lamin levels leading to dilated cardiomyopathy, which presumably arose independently from conduction system disease.

We identified a human mutation that causes dilated cardiomyopathy and heart failure preceded by sensorineural hearing loss (SNHL). Unlike previously described mutations causing dilated cardiomyopathy that affect structural proteins, this mutation deletes 4,846 bp of the human transcriptional coactivator gene EYA4. To elucidate the roles of eya4 in heart function, we studied zebrafish embryos injected with antisense morpholino oligonucleotides. Attenuated eya4 transcript levels produced morphologic and hemodynamic features of heart failure. To determine why previously described mutated EYA4 alleles1,2 cause SNHL without heart disease, we examined biochemical interactions of mutant Eya4 peptides. Eya4 peptides associated with SNHL, but not the shortened 193–amino acid peptide associated with dilated cardiomyopathy and SNHL, bound wild-type Eya4 and associated with Six proteins. These data define unrecognized and crucial roles for Eya4-Six–mediated transcriptional regulation in normal heart function.