Caveolin-3 and Caveolae regulate ventricular repolarization.

RATIONALE: Flask-shaped invaginations of the cardiomyocyte sarcolemma called caveolae require the structural protein caveolin-3 (Cav-3) and host a variety of ion channels, transporters, and signaling molecules. Reduced Cav-3 expression has been reported in models of heart failure, and variants in CAV3 have been associated with the inherited long-QT arrhythmia syndrome. Yet, it remains unclear whether alterations in Cav-3 levels alone are sufficient to drive aberrant repolarization and increased arrhythmia risk. OBJECTIVE: To determine the impact of cardiac-specific Cav-3 ablation on the electrophysiological properties of the adult mouse heart. METHODS AND RESULTS: Cardiac-specific, inducible Cav3 homozygous knockout (Cav-3KO) mice demonstrated a marked reduction in Cav-3 expression by Western blot and loss of caveolae by electron microscopy. However, there was no change in macroscopic cardiac structure or contractile function. The QTc interval was increased in Cav-3KO mice, and there was an increased propensity for ventricular arrhythmias. Ventricular myocytes isolated from Cav-3KO mice exhibited a prolonged action potential duration (APD) that was due to reductions in outward potassium currents (Ito, Iss) and changes in inward currents including slowed inactivation of ICa,L and increased INa,L. Mathematical modeling demonstrated that the changes in the studied ionic currents were adequate to explain the prolongation of the mouse ventricular action potential. Results from human iPSC-derived cardiomyocytes showed that shRNA knockdown of Cav-3 similarly prolonged APD. CONCLUSION: We demonstrate that Cav-3 and caveolae regulate cardiac repolarization and arrhythmia risk via the integrated modulation of multiple ionic currents.

Induced cardiac progenitor cells repopulate decellularized mouse heart scaffolds and differentiate to generate cardiac tissue.

Native myocardium has limited regenerative potential post injury. Advances in lineage reprogramming have provided promising cellular sources for regenerative medicine in addition to research applications. Recently we have shown that adult mouse fibroblasts can be reprogrammed to expandable, multipotent, induced cardiac progenitor cells (iCPCs) by employing forced expression of five cardiac factors along with activation of canonical Wnt and JAK/STAT signaling. Here we aim to further characterize iCPCs by highlighting their safety, ease of attainability, and functionality within a three-dimensional cardiac extracellular matrix scaffold. Specifically, iCPCs did not form teratomas in contrast to embryonic stem cells when injected into immunodeficient mice. iCPC reprogramming was achieved in wild type mouse fibroblasts without requiring a cardiac-specific reporter, solely utilizing morphological changes to identify, clonally isolate, and expand iCPCs, thus increasing the versatility of this technology. iCPCs also show the ability to repopulate decellularized native heart scaffolds and differentiated into organized structures containing cardiomyocytes, smooth muscle, and endothelial cells. Optical mapping of recellularized scaffolds shows field-stimulated calcium transients that propagate across islands of reconstituted tissue and bipolar local stimulation demonstrates cell-cell coupling within scaffolds. Overall, iCPCs provide a readily attainable, scalable, safe, and functional cell source for a variety of application including drug discovery, disease modeling, and regenerative therapy.

Pediatric Dilated Cardiomyopathy-Associated LRRC10 (Leucine-Rich Repeat-Containing 10) Variant Reveals LRRC10 as an Auxiliary Subunit of Cardiac L-Type Ca2+ Channels.

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 Ca2+ channel (Cav1.2, ß2CN2, and α2δ subunits) in HEK293 cells resulted in a significant ≈0.5-fold decrease in ICa,L at 0 mV, in contrast to the ≈1.4-fold increase in ICa,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 Cav1.2. LRRC10 coexpression with Cav1.2 in the absence of auxiliary ß2CN2 and α2δ subunits revealed coassociation of Cav1.2 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 ICa,L, and coimmunoprecipitation experiments confirmed association between LRRC10 and the Cav1.2 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 Ca2+ channels and exert dramatically different effects on channel gating, providing a potential link to DCM.

Lineage Reprogramming of Fibroblasts into Proliferative Induced Cardiac Progenitor Cells by Defined Factors.

Several studies have reported reprogramming of fibroblasts into induced cardiomyocytes; however, reprogramming into proliferative induced cardiac progenitor cells (iCPCs) remains to be accomplished. Here we report that a combination of 11 or 5 cardiac factors along with canonical Wnt and JAK/STAT signaling reprogrammed adult mouse cardiac, lung, and tail tip fibroblasts into iCPCs. The iCPCs were cardiac mesoderm-restricted progenitors that could be expanded extensively while maintaining multipotency to differentiate into cardiomyocytes, smooth muscle cells, and endothelial cells in vitro. Moreover, iCPCs injected into the cardiac crescent of mouse embryos differentiated into cardiomyocytes. iCPCs transplanted into the post-myocardial infarction mouse heart improved survival and differentiated into cardiomyocytes, smooth muscle cells, and endothelial cells. Lineage reprogramming of adult somatic cells into iCPCs provides a scalable cell source for drug discovery, disease modeling, and cardiac regenerative therapy.