Abstract 15700: L-type Calcium Channels Localization and Function are Affected by Microdomain Remodelling in Human Failing Cardiomyocytes

Abstract

Introduction: L-type calcium channels (LTCCs) are essential in determining the electrical and mechanical properties of the cardiac muscle. LTCCs are located in specialized membrane microdomains in cardiac cells. Disruption of their microdomain localization in heart failure may contribute to the pathophysiology of cardiac disease. Methods: We studied isolated ventricular cardiomyocytes from patients with dilated cardiomyopathy with or without Left Ventricular Assistance Device (DCM or DCM+LVAD) versus those with normal ventricular function (nonfailing). Super-resolution scanning patch clamp provided topography images of the cell surface which enabled us to record LTCC current in distinct cellular compartments (T-tubules openings and areas between them, crests). Confocal microscopy was used to score T-tubular density and regularity in Di-8-ANEPPS stained cells. Expression of β2 subunit of LTCC was detected by immunofluorescence, Western blot and qPCR. Results: In failing cells T-tubular density is lower than in normal. The loss of cellular micro-architecture in failing cardiomyocytes leads to a redistribution of functional LTCCs from their usual location (T-tubules) to non-native crests of the sarcolemma. These relocated LTCCs in the crest of DCM cardiomyocytes have increased open probability and lower amplitude as compared with the channels located on the T-tubules. Interestingly, in cardiomyocytes isolated from patients with LVAD the open probability of LTCC is reduced dramatically and the amplitude recovers to the normal level, as does the T-tubular density. Immunostaining of DCM cardiomyocytes shows an increased amount of LTCC β2 subunit in the crest, and Western blot/qPCR shows more abundant expression of LTCC β2 subunit. Conclusion: In DCM cardiomyocytes a considerable amount of LTCCs improperly located on the crest microdomain display a pathologically high activity; this study suggests that the increased expression of LTCC β2-subunit could be responsible. Although LVAD cannot reverse the remodelling in DCM cardiomyocytes it helps to restore part of the microarchitecture of the cells and to normalize the function of the LTCCs.