Kamp, Timothy J.

Leucine-rich repeat containing (LRR) proteins facilitate protein-protein interactions critical in a number of cellular functions including ion channel regulation. Leucine-rich repeat containing protein 10 (LRRC10), a cardiac-specific protein expressed in zebrafish, mice and humans, is essential for normal cardiac physiology with loss of Lrrc10 in Lrrc10 -/- mice resulting in dilated cardiomyopathy (DCM). However, the mechanism by which LRRC10 contributes to DCM is unknown. Further, the functional role of LRRC10 in the regulation of ion channels in the heart has not been explored. Here we recorded L-type Ca 2+ channel currents (I Ca,L ) from isolated ventricular myocytes of WT and Lrrc10 -/- mice, demonstrating a significant reduction in I Ca,L in Lrrc10 -/- myocytes (-4.9 ± 0.19 pA/pF, n=10) compared to WT (-6.5 ± 0.24 pA/pF, n=8). Co-immunoprecipitation (co-IP) experiments showed an association between LRRC10 and Ca v 1.2 in WT, but not Lrrc10 -/- mouse lysates, with immunocytochemistry studies further demonstrating colocalization of LRRC10 and Ca v 1.2 in isolated ventricular myocytes from wild-type (WT) mice. Additionally, whole exome sequencing revealed a novel homozygous recessive missense variant in LRRC10 , I195T, found in a pediatric DCM patient. To determine whether the I195T variant impacts Ca v 1.2 L-type Ca 2+ channels, whole-cell patch clamp experiments were performed using HEK293 cells transiently expressing the L-type Ca 2+ channel complex (LTCC) alone or with WT or I195T LRRC10. These electrophysiology experiments demonstrated a significant increase in I Ca,L with WT LRRC10 coexpression (-81 ± 5.3 pA/pF, n=12), but a decrease in I Ca,L when I195T (-18.2 ± 3.3 pA/pF, n=9) was coexpressed compared to the LTCC alone (-34.1 ± 2.2 pA/pF, n=17). Parallel surface biotinylation experiments demonstrated that WT and I195T LRRC10 did not alter L-type Ca 2+ channel expression on the plasma membrane, while co-IP experiments using lysates prepared from the transiently transfected HEK293 cells showed the association between Ca v 1.2 and I195T LRRC10 variant. Overall, these findings newly identify LRRC10 as a cardiac-specific component of the Ca v 1.2 macromolecular complex and demonstrate dysregulation of I Ca,L by the DCM associated I195T LRRC10 variant.

Key points Mutations in the caveolae scaffolding protein, caveolin‐3 (Cav3), have been linked to the long QT type 9 inherited arrhythmia syndrome (LQT9) and the cause of underlying action potential duration prolongation is incompletely understood. In the present study, we show that LQT9 Cav3 mutations, F97C and S141R, cause mutation‐specific gain of function effects on Cav1.2‐encoded L‐type Ca2+ channels responsible for ICa,L and also cause loss of function effects on heterologously expressed Kv4.2 and Kv4.3 channels responsible for Ito. A computational model of the human ventricular myocyte action potential suggests that the major ionic current change causing action potential duration prolongation in the presence of Cav3‐F97C is the slowly inactivating ICa,L but, for Cav3‐S141R, both increased ICa,L and increased late Na+ current contribute equally to action potential duration prolongation. Overall, the LQT9 Cav3‐F97C and Cav3‐S141R mutations differentially impact multiple ionic currents, highlighting the complexity of Cav3 regulation of cardiac excitability and suggesting mutation‐specific therapeutic approaches. AbstractMutations in the CAV3 gene encoding caveolin‐3 (Cav3), a scaffolding protein integral to caveolae in cardiomyocytes, have been associated with the congenital long‐QT syndrome (LQT9). Initial studies demonstrated that LQT9‐associated Cav3 mutations, F97C and S141R, increase late sodium current as a potential mechanism to prolong action potential duration (APD) and cause LQT9. Whether these Cav3 LQT9 mutations impact other caveolae related ion channels remains unknown. We used the whole‐cell, patch clamp technique to characterize the effect of Cav3‐F97C and Cav3‐S141R mutations on heterologously expressed Cav1.2+Cavβ2cN4 channels, as well as Kv4.2 and Kv4.3 channels, in HEK 293 cells. Expression of Cav3‐S141R increased ICa,L density without changes in gating properties, whereas expression of Cav3‐F97C reduced Ca2+‐dependent inactivation of ICa,L without changing current density. The Cav3‐F97C mutation reduced current density and altered the kinetics of IKv4.2 and IKv4.3 and also slowed recovery from inactivation. Cav3‐S141R decreased current density and also slowed activation kinetics and recovery from inactivation of IKv4.2 but had no effect on IKv4.3. Using the O'Hara–Rudy computational model of the human ventricular myocyte action potential, the Cav3 mutation‐induced changes in Ito are predicted to have negligible effect on APD, whereas blunted Ca2+‐dependent inactivation of ICa,L by Cav3‐F97C is predicted to be primarily responsible for APD prolongation, although increased ICa,L and late INa by Cav3‐S141R contribute equally to APD prolongation. Thus, LQT9 Cav3‐associated mutations, F97C and S141R, produce mutation‐specific changes in multiple ionic currents leading to different primary causes of APD prolongation, which suggests the use of mutation‐specific therapeutic approaches in the future.

Background Genetic causes of dilated cardiomyopathy (DCM) are incompletely understood. LRRC10 (leucine‐rich repeat–containing 10) is a cardiac‐specific protein of unknown function. Heterozygous mutations in LRRC10 have been suggested to cause DCM, and deletion of Lrrc10 in mice results in DCM. Methods and Results Whole‐exome sequencing was carried out on a patient who presented at 6 weeks of age with DCM and her unaffected parents, filtering for rare, deleterious, recessive, and de novo variants. Whole‐exome sequencing followed by trio‐based filtering identified a homozygous recessive variant in LRRC10 , I195T. Coexpression of I195T LRRC10 with the L‐type Ca 2+ channel (Ca v 1.2, β 2CN2 , and α 2 δ subunits) in HEK293 cells resulted in a significant ≈0.5‐fold decrease in I Ca,L at 0 mV, in contrast to the ≈1.4‐fold increase in I Ca,L by coexpression of LRRC10 (n=9–12, P <0.05). Coexpression of LRRC10 or I195T LRRC10 did not alter the surface membrane expression of Ca v 1.2. LRRC10 coexpression with Ca v 1.2 in the absence of auxiliary β 2CN2 and α 2 δ subunits revealed coassociation of Ca v 1.2 and LRRC10 and a hyperpolarizing shift in the voltage dependence of activation (n=6–9, P <0.05). Ventricular myocytes from Lrrc10 −/− mice had significantly smaller I Ca,L , and coimmunoprecipitation experiments confirmed association between LRRC10 and the Ca v 1.2 subunit in mouse hearts. Conclusions Examination of a patient with DCM revealed homozygosity for a previously unreported LRRC10 variant: I195T. Wild‐type and I195T LRRC10 function as cardiac‐specific subunits of L‐type Ca 2+ channels and exert dramatically different effects on channel gating, providing a potential link to DCM.

We previously reported that the cardiomyocyte-specific leucine-rich repeat containing protein (LRRC)10 has critical functions in the mammalian heart. In the present study, we tested the role of LRRC10 in the response of the heart to biomechanical stress by performing transverse aortic constriction on Lrrc10-null ( Lrrc10−/−) mice. Mild pressure overload induced severe cardiac dysfunction and ventricular dilation in Lrrc10−/−mice compared with control mice. In addition to dilation and cardiomyopathy, Lrrc10−/−mice showed a pronounced increase in heart weight with pressure overload stimulation and a more dramatic loss of cardiac ventricular performance, collectively suggesting that the absence of LRRC10 renders the heart more disease prone with greater hypertrophy and structural remodeling, although rates of cardiac fibrosis and myocyte dropout were not different from control mice. Lrrc10−/−cardiomyocytes also exhibited reduced contractility in response to β-adrenergic stimulation, consistent with loss of cardiac ventricular performance after pressure overload. We have previously shown that LRRC10 interacts with actin in the heart. Here, we show that His150of LRRC10 was required for an interaction with actin, and this interaction was reduced after pressure overload, suggesting an integral role for LRRC10 in the response of the heart to mechanical stress. Importantly, these experiments demonstrated that LRRC10 is required to maintain cardiac performance in response to pressure overload and suggest that dysregulated expression or mutation of LRRC10 may greatly sensitize human patients to more severe cardiac disease in conditions such as chronic hypertension or aortic stenosis.

Background— Long QT syndrome (LQTS) is the most common cardiac channelopathy with 15 elucidated LQTS-susceptibility genes. Approximately 20% of LQTS cases remain genetically elusive. Methods and Results— We combined whole-exome sequencing and bioinformatic/systems biology to identify the pathogenic substrate responsible for nonsyndromic, genotype-negative, autosomal dominant LQTS in a multigenerational pedigree, and we established the spectrum and prevalence of variants in the elucidated gene among a cohort of 102 unrelated patients with “genotype-negative/phenotype-positive” LQTS. Whole-exome sequencing was used on 3 members within a genotype-negative/phenotype-positive family. Genomic triangulation combined with bioinformatic tools and ranking algorithms led to the identification of a CACNA1C mutation. This mutation, Pro857Arg-CACNA1C, cosegregated with the disease within the pedigree, was ranked by 3 disease-network algorithms as the most probable LQTS-susceptibility gene and involves a conserved residue localizing to the proline, gltamic acid, serine, and threonine (PEST) domain in the II-III linker. Functional studies reveal that Pro857Arg-CACNA1C leads to a gain of function with increased I Ca,L and increased surface membrane expression of the channel compared to wild type. Subsequent mutational analysis identified 3 additional variants within CACNA1C in our cohort of 102 unrelated cases of genotype-negative/phenotype-positive LQTS. Two of these variants also involve conserved residues within Ca v 1.2’s PEST domain. Conclusions— This study provides evidence that coupling whole-exome sequencing and bioinformatic/systems biology is an effective strategy for the identification of potential disease-causing genes/mutations. The identification of a functional CACNA1C mutation cosegregating with disease in a single pedigree suggests that CACNA1C perturbations may underlie autosomal dominant LQTS in the absence of Timothy syndrome.

Background— Congenital long-QT syndrome (LQTS) is a primary arrhythmogenic syndrome stemming from perturbed cardiac repolarization. LQTS, which affects ≈1 in 3000 persons, is 1 of the most common causes of autopsy-negative sudden death in the young. Since the sentinel discovery of cardiac channel gene mutations in LQTS in 1995, hundreds of mutations in 8 LQTS susceptibility genes have been identified. All 8 LQTS genotypes represent primary cardiac channel defects (ie, ion channelopathy) except LQT4, which is a functional channelopathy because of mutations in ankyrin-B. Approximately 25% of LQTS remains unexplained pathogenetically. We have pursued a “final common pathway” hypothesis to elicit novel LQTS-susceptibility genes. With the recent observation that the LQT3-associated, SCN5A -encoded cardiac sodium channel localizes in caveolae, which are known membrane microdomains whose major component in the striated muscle is caveolin-3, we hypothesized that mutations in caveolin-3 may represent a novel pathogenetic mechanism for LQTS. Methods and Results— Using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing, we performed open reading frame/splice site mutational analysis on CAV3 in 905 unrelated patients referred for LQTS genetic testing. CAV3 mutations were engineered by site-directed mutagenesis and the molecular phenotype determined by transient heterologous expression into cell lines that stably express the cardiac sodium channel hNa v 1.5. We identified 4 novel mutations in CAV3 -encoded caveolin-3 that were absent in >1000 control alleles. Electrophysiological analysis of sodium current in HEK293 cells stably expressing hNa v 1.5 and transiently transfected with wild-type and mutant caveolin-3 demonstrated that mutant caveolin-3 results in a 2- to 3-fold increase in late sodium current compared with wild-type caveolin-3. Our observations are similar to the increased late sodium current associated with LQT3-associated SCN5A mutations. Conclusions— The present study reports the first CAV3 mutations in subjects with LQTS, and we provide functional data demonstrating a gain-of-function increase in late sodium current.