Noonan and LEOPARD syndromes are developmental disorders with overlapping features, including cardiac abnormalities, short stature and facial dysmorphia. Increased RAS signaling owing to PTPN11, SOS1 and KRAS mutations causes approximately 60% of Noonan syndrome cases, and PTPN11 mutations cause 90% of LEOPARD syndrome cases. Here, we report that 18 of 231 individuals with Noonan syndrome without known mutations (corresponding to 3% of all affected individuals) and two of six individuals with LEOPARD syndrome without PTPN11 mutations have missense mutations in RAF1, which encodes a serine-threonine kinase that activates MEK1 and MEK2. Most mutations altered a motif flanking Ser259, a residue critical for autoinhibition of RAF1 through 14-3-3 binding. Of 19 subjects with a RAF1 mutation in two hotspots, 18 (or 95%) showed hypertrophic cardiomyopathy (HCM), compared with the 18% prevalence of HCM among individuals with Noonan syndrome in general. Ectopically expressed RAF1 mutants from the two HCM hotspots had increased kinase activity and enhanced ERK activation, whereas non-HCM-associated mutants were kinase impaired. Our findings further implicate increased RAS signaling in pathological cardiomyocyte hypertrophy.

There has been significant progress in our understanding of the molecular mechanisms by which calcium (Ca2+) ions mediate various types of cardiac arrhythmias. A growing list of inherited gene defects can cause potentially lethal cardiac arrhythmia syndromes, including catecholaminergic polymorphic ventricular tachycardia, congenital long QT syndrome, and hypertrophic cardiomyopathy. In addition, acquired deficits of multiple Ca2+-handling proteins can contribute to the pathogenesis of arrhythmias in patients with various types of heart disease. In this review article, we will first review the key role of Ca2+ in normal cardiac function - in particular, excitation-contraction coupling and normal electrical rhythms. The functional involvement of Ca2+ in distinct arrhythmia mechanisms will be discussed, followed by various inherited arrhythmia syndromes caused by mutations in Ca2+-handling proteins. Finally, we will discuss how changes in the expression of regulation of Ca2+ channels and transporters can cause acquired arrhythmias, and how these mechanisms might be targeted for therapeutic purposes.

Background-Excitation-contraction coupling in striated muscle requires proper communication of plasmalemmal voltage-activated Ca 2ϩ channels and Ca 2ϩ 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 2ϩ -induced Ca 2ϩ 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 excitationcontraction 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 Ca 2ϩ release channel inactivation, suggesting a novel functional role for JPH2 in regulating intracellular Ca 2ϩ 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 Ca 2ϩ release in the heart. (Circulation. 2011;123:979-988.)Key Words: calcium Ⅲ excitation Ⅲ heart failure Ⅲ junctophilin Ⅲ sarcoplasmic reticulum E xcitation-contraction (EC) coupling is the fundamental mechanism by which depolarization of the voltage-gated Ca 2ϩ channels (VGCCs) in the plasmalemma triggers a much greater release of Ca 2ϩ from the sarcoplasmic reticulum (SR) via type 2 ryanodine receptors (RyR2), a process known as Ca 2ϩ -induced Ca 2ϩ release (CICR). 1 This Ca 2ϩ release amplification depends on the organization of VGCC and RyR2 within junctional membrane complexes (JMCs), also known as calcium release units. 2 Disruption of JMC structure, as seen in heart failure, profoundly affects CICR and thus cardiac muscle contractility. 3 Clinical Perspective on p 988The molecular mechanisms involved in organizing Ca 2ϩ channels within the JMC remain poorly understood. One family of proteins, known as junctophilins (JPHs), has been proposed to provide a structural bridge between the plasmalemma and SR, thereby potentially ensuring approximation of VGCC and RyR2. 4 Junctophilin-2 (JPH2) is the major cardiac isoform among the 4 JPH isoforms, which are expressed within JMCs of all excitable cell types. 5 JPH proteins comprise 8 N-terminal "membrane occupation and recognition nexus" domains, a space-spanning ␣-helix, and a C-terminal transmembrane domain. The membrane occupation and recognition nexus domains mediate binding to the plasmalemma, and the hydrophobic transmembrane domain is anchored into ...