Faculty Bibliography

Cardiomyocytes are subjected to the intense mechanical stress and metabolic demands of the beating heart. It is unclear whether these cells, which are long-lived and rarely renew, manage to preserve homeostasis on their own. While analyzing macrophages lodged within the healthy myocardium, we discovered that they actively took up material, including mitochondria, derived from cardiomyocytes. Cardiomyocytes ejected dysfunctional mitochondria and other cargo in dedicated membranous particles reminiscent of neural exophers, through a process driven by the cardiomyocyte's autophagy machinery that was enhanced during cardiac stress. Depletion of cardiac macrophages or deficiency in the phagocytic receptor Mertk resulted in defective elimination of mitochondria from the myocardial tissue, activation of the inflammasome, impaired autophagy, accumulation of anomalous mitochondria in cardiomyocytes, metabolic alterations, and ventricular dysfunction. Thus, we identify an immune-parenchymal pair in the murine heart that enables transfer of unfit material to preserve metabolic stability and organ function.

Background: Genetic variants in calsequestrin-2 (CASQ2) cause an autosomal recessive form of catecholaminergic polymorphic ventricular tachycardia (CPVT), though isolated reports have identified arrhythmic phenotypes among heterozygotes. Improved insight into the inheritance patterns, arrhythmic risks, and molecular mechanisms of CASQ2-CPVT was sought through an international multi-center collaboration. Methods: Genotype-phenotype segregation in CASQ2-CPVT families was assessed, and the impact of genotype on arrhythmic risk was evaluated using Cox regression models. Putative dominant CASQ2 missense variants and the established recessive CASQ2-p.R33Q variant were evaluated using oligomerization assays and their locations mapped to a recent CASQ2 filament structure. Results: A total of 112 individuals, including 36 CPVT probands (24 homozygotes/compound heterozygotes and 12 heterozygotes) and 76 family members possessing at least one presumed pathogenic CASQ2 variant, were identified. Among CASQ2 homozygotes and compound heterozygotes, clinical penetrance was 97.1% and 26 of 34 (76.5%) individuals had experienced a potentially fatal arrhythmic event with a median age of onset of 7 years (95% CI: 6-11). Fifty-one of 66 CASQ2 heterozygous family members had undergone clinical evaluation and 17/51 (33.3%) met diagnostic criteria for CPVT. Relative to CASQ2 heterozygotes, CASQ2 homozygote/compound heterozygote genotype status in probands was associated with a 3.2-fold (95% confidence intervals [CI]: 1.3-8.0, p=0.013) increased hazard of a composite of cardiac syncope, aborted cardiac arrest, and sudden cardiac death, but a 38.8-fold (95% CI: 5.6-269.1, p<0.001) increased hazard in genotype positive family members. In vitro turbidity assays revealed that p.R33Q and all 6 candidate dominant CASQ2 missense variants evaluated exhibited filamentation defects, but only p.R33Q convincingly failed to dimerize. Structural analysis revealed that 3 of these 6 putative dominant negative missense variants localized to an electronegative pocket considered critical for back-to-back binding of dimers. Conclusions: This international multi-center study of CASQ2-CPVT redefines its heritability and confirms that pathogenic heterozygous CASQ2 variants may manifest with a CPVT phenotype, indicating a need to clinically screen these individuals. A dominant mode of inheritance appears intrinsic to certain missense variants owing to their location and function within the CASQ2 filament structure.

Background - In cardiac gene therapy to improve contractile function, achieving gene expression in the majority of cardiac myocytes is essential. In preventing cardiac arrhythmias, however, this goal may not be as important, since transduction efficiencies as low as 40% suppressed ventricular arrhythmias in genetically-modified mice with polymorphic catecholaminergic ventricular tachycardia (CPVT). Methods - Using computational modeling, we simulated 1D, 2D and 3D tissue under a variety of conditions to test the ability of genetically-engineered non-arrhythmogenic "stabilizer cells" to suppress triggered activity (TA) due to delayed (DAD) or early (EAD) afterdepolarizations. Results - Due to source-sink relationships in cardiac tissue, a minority (20-50%) of randomly distributed "stabilizer" cells engineered to be non-arrhythmogenic can suppress the ability of arrhythmogenic cells to generate DAD- and EAD-related arrhythmias. Stabilizer cell gene therapy strategy can be designed to correct a specific arrhythmogenic mutation, as in the CPVT mice studies, or more generally to suppress DADs or EADs from any cause by overexpressing the inward rectifier K channel Kir2.1 in stabilizer cells. Conclusions - This promising antiarrhythmic strategy warrants further testing in experimental models to evaluate its clinical potential.

Background: Long QT syndrome (LQTS) is a rare genetic disorder and a major preventable cause of sudden cardiac death in the young. A causal rare genetic variant with large effect size is identified in up to 80% of probands (genotype positive) and cascade family screening shows incomplete penetrance of genetic variants. Furthermore, a proportion of cases meeting diagnostic criteria for LQTS remain genetically elusive despite genetic testing of established genes (genotype negative). These observations raise the possibility that common genetic variants with small effect size contribute to the clinical picture of LQTS. This study aimed to characterize and quantify the contribution of common genetic variation to LQTS disease susceptibility. Methods: We conducted genome-wide association studies (GWAS) followed by transethnic meta-analysis in 1,656 unrelated LQTS patients of European or Japanese ancestry and 9,890 controls to identify susceptibility single nucleotide polymorphisms (SNPs). We estimated the SNP heritability (h²_SNP) of LQTS and tested the genetic correlation between LQTS susceptibility and other cardiac traits. Furthermore, we tested the aggregate effect of the 68 SNPs previously associated with QTc in the general population using a polygenic risk score (PRS_QT). Results: Genome-wide association analysis identified three loci associated with LQTS at genome-wide statistical significance (P<5x10^-8) near NOS1AP, KCNQ1 and KLF12, and one missense variant in KCNE1 (p.Asp85Asn) at the suggestive threshold (P<10^-6). Heritability analyses showed that ~15% of variance in overall LQTS susceptibility was attributable to common genetic variation (h²_SNP 0.148; standard error [SE] 0.019). LQTS susceptibility showed a strong genome-wide genetic correlation with the QT interval in the general population (r_g=0.40, P=3.2x10^-3). PRS_QT was greater in LQTS cases compared to controls (P<10^-13), and notably, among LQTS patients PRS_QT was greater in genotype negative compared to genotype positive patients (P<0.005). Conclusions: This work establishes an important role for common genetic variation in susceptibility to LQTS. We demonstrate overlap between genetic control of the QT interval in the general population and genetic factors contributing to LQTS susceptibility. Using polygenic risk score analyses aggregating common genetic variants that modulate the QT interval in the general population, we provide evidence for a polygenic architecture in genotype negative LQTS.

Heteroplasmy, multiple variants of mitochondrial DNA (mtDNA) in the same cytoplasm, may be naturally generated by mutations but is counteracted by a genetic mtDNA bottleneck during oocyte development. Engineered heteroplasmic mice with nonpathological mtDNA variants reveal a nonrandom tissue-specific mtDNA segregation pattern, with few tissues that do not show segregation. The driving force for this dynamic complex pattern has remained unexplained for decades, challenging our understanding of this fundamental biological problem and hindering clinical planning for inherited diseases. Here, we demonstrate that the nonrandom mtDNA segregation is an intracellular process based on organelle selection. This cell type-specific decision arises jointly from the impact of mtDNA haplotypes on the oxidative phosphorylation (OXPHOS) system and the cell metabolic requirements and is strongly sensitive to the nuclear context and to environmental cues.

Background: Mutations in desmoplakin (DSP), the primary force transducer between cardiac desmosomes and intermediate filaments, cause an arrhythmogenic form of cardiomyopathy that has been variably associated with arrhythmogenic right ventricular cardiomyopathy (ARVC). Clinical correlates of DSP cardiomyopathy have been limited to small case series. Methods: Clinical and genetic data were collected on 107 patients with pathogenic DSP mutations and 81 patients with pathogenic PKP2 mutations as a comparison cohort. A composite outcome of severe ventricular arrhythmia was assessed. Results:DSP and PKP2 cohorts included similar proportions of probands (41% vs. 42%) and patients with truncating mutations (98% vs. 100%). Left ventricular (LV) predominant cardiomyopathy was exclusively present among DSP patients (55% vs. 0% for PKP2, p<0.001), whereas right ventricular (RV) cardiomyopathy was present in only 14% of DSP patients vs. 40% for PKP2 (p<0.001). ARVC diagnostic criteria had poor sensitivity for DSP cardiomyopathy. LV late gadolinium enhancement (LGE) was present in a primarily subepicardial distribution in 40% of DSP patients (23/57 with MRIs). LV LGE occurred with normal LV systolic function in 35% (8/23) of DSP patients. Episodes of acute myocardial injury (chest pain with troponin elevation and normal coronary angiography) occurred in 15% of DSP patients and were strongly associated with LV LGE (90%), even in cases of acute myocardial injury with normal ventricular function (4/5, 80% with LGE). In 4 DSP cases with ¹⁸F-fluorodeoxyglucose positron emission tomography scans, acute LV myocardial injury was associated with myocardial inflammation misdiagnosed initially as cardiac sarcoidosis or myocarditis. LVEF <55% was strongly associated with severe ventricular arrhythmias for DSP cases (p<0.001, sensitivity 85%, specificity 53%). RVEF <45% was associated with severe arrhythmias for PKP2 cases (p<0.001) but was poorly associated for DSP cases (p=0.8). Frequent PVCs were common among patients with severe arrhythmias for both DSP (80%) and PKP2 (91%) groups (p=NS). Conclusions:DSP cardiomyopathy is a distinct form of arrhythmogenic cardiomyopathy characterized by episodic myocardial injury, left ventricular fibrosis that precedes systolic dysfunction, and a high incidence of ventricular arrhythmias. A genotype specific approach for diagnosis and risk stratification should be used.

Background Atrial fibrillation (AF) is a comorbidity associated with heart failure and catecholaminergic polymorphic ventricular tachycardia. Despite the Ca²⁺-dependent nature of both of these pathologies, AF often responds to Na⁺ channel blockers. We investigated how targeting interdependent Na⁺/Ca²⁺ dysregulation might prevent focal activity and control AF. Methods and Results We studied AF in 2 models of Ca²⁺-dependent disorders, a murine model of catecholaminergic polymorphic ventricular tachycardia and a canine model of chronic tachypacing-induced heart failure. Imaging studies revealed close association of neuronal-type Na⁺ channels (nNa_v) with ryanodine receptors and Na⁺/Ca²⁺ exchanger. Catecholamine stimulation induced cellular and in vivo atrial arrhythmias in wild-type mice only during pharmacological augmentation of nNa_v activity. In contrast, catecholamine stimulation alone was sufficient to elicit atrial arrhythmias in catecholaminergic polymorphic ventricular tachycardia mice and failing canine atria. Importantly, these were abolished by acute nNa_v inhibition (tetrodotoxin or riluzole) implicating Na⁺/Ca²⁺ dysregulation in AF. These findings were then tested in 2 nonrandomized retrospective cohorts: an amyotrophic lateral sclerosis clinic and an academic medical center. Riluzole-treated patients adjusted for baseline characteristics evidenced significantly lower incidence of arrhythmias including new-onset AF, supporting the preclinical results. Conclusions These data suggest that nNa_Vs mediate Na⁺-Ca²⁺ crosstalk within nanodomains containing Ca²⁺ release machinery and, thereby, contribute to AF triggers. Disruption of this mechanism by nNa_v inhibition can effectively prevent AF arising from diverse causes.