Multiplexed measurement of cell type-specific calcium kinetics using high-content image analysis combined with targeted gene disruption.

Kinetic analysis of intracellular calcium (Ca2+) in cardiomyocytes is commonly used to determine the pathogenicity of genetic mutations identified in patients with dilated cardiomyopathy (DCM). Conventional methods for measuring Ca2+ kinetics target whole-well cultured cardiomyocytes and therefore lack information concerning individual cells. Results are also affected by heterogeneity in cell populations. Here, we developed an analytical method using CRISPR/Cas9 genome editing combined with high-content image analysis (HCIA) that links cell-by-cell Ca2+ kinetics and immunofluorescence images in thousands of cardiomyocytes at a time. After transfecting cultured mouse cardiomyocytes that constitutively express Cas9 with gRNAs, we detected a prolonged action potential duration specifically in Serca2a-depleted ventricular cardiomyocytes in mixed culture. To determine the phenotypic effect of a frameshift mutation in PKD1 in a patient with DCM, we introduced the mutation into Cas9-expressing cardiomyocytes by gRNA transfection and found that it decreases the expression of PKD1-encoded PC1 protein that co-localizes specifically with Serca2a and L-type voltage-gated calcium channels. We also detected the suppression of Ca2+ amplitude in ventricular cardiomyocytes with decreased PC1 expression in mixed culture. Our HCIA method provides comprehensive kinetic and static information on individual cardiomyocytes and allows the pathogenicity of mutations to be determined rapidly.

Old-Age Onset Progressive Cardiac Contractile Dysfunction in a Patient with Polycystic Kidney Disease Harboring a PKD1 Frameshift Mutation.

A 70-year-old man with dyspnea was admitted to our department and received standard therapy for recurrent heart failure. He was diagnosed with polycystic kidney disease (PKD) in his thirties and received hemodialysis for 4 years before undergoing renal transplantation at age 45. Although his left ventricular ejection fraction (LVEF) was preserved in his 50s, LVEF decreased progressively from 61% to 24%, while left ventricular diastolic dimension (LVDd) increased from 54 mm to 65 mm between 63 and 69 years of age. Right ventricular endomyocardial biopsy demonstrated myocardial disarray and interstitial fibrosis. Genetic analysis identified a heterozygous frameshift mutation in PKD1, which encodes polycystin-1, a major causative gene of PKD. We detected PKD1 protein expression in myocardial tissue by immunostaining. Recent epidemiological studies and animal models have clarified the pathological correlation between ventricular contractile dysfunction and PKD1 function. Here, we present a case of old-age onset progressive cardiac contractile dysfunction with a PKD1 gene mutation.