Introduction:
Cardiovascular disease (CVD) is a leading public health problem in the UK, with over 7 million people affected and at a cost of more than 150,000 deaths and £10 billion annually. Such a major cause of morbidity, mortality and economic burden reveals a huge medical need for novel therapeutic targets to treat CVD. It is well established that stimulation of CaV1.2 voltage-gated Ca2+ channels (VGCCs) plays a fundamental role in cardiac cell function, regulating both rate and rhythm and contractility of the heartbeat. Therefore, understanding novel regulatory mechanisms of CaV1.2 VGCCs may reveal new targets to treat CVD such as heart failure and arrhythmogenesis. We recently showed that myristoylated alanine-rich C kinase substrate (MARCKS) acts as phosphatidylinositol 4,5-bisphosphate (PIP2) buffer to regulate the activity of CaV1.2 VGGCs and vasoconstriction of mesenteric arteries (Jahan et al. 2020).
Aim: In the present study, we have investigated whether similar mechanisms are present in cardiomyocytes.
Methods:
All animal procedures were carried out in accordance with guidelines laid down by St George's, University of London Animal Welfare Committee and conform with the principles and regulations described by the Service Project Licence: 70/8512 Freshly isolated heart lysates and cardiomyocytes from hearts of male Wistar rats were studied through a wide range of techniques including western blotting, immunocytochemistry, proximity ligation assay (PLA), and whole-cell patch clamp electrophysiology.
Results:
In western blot studies, expression levels of MARCKS, CaV1.2, and PIP2 were similar in right atrium, right ventricle, left atrium, and left ventricle (n= 7, p˃0.05, one-way ANOVA). When heart samples were incubated with wortmannin (20 μM), PIP2 levels were significantly supressed in all heart chambers (P<0.0001, one-way ANOVA). The functionality of MARCKS on VGCCs was then investigated using the MARCK activator, MANS peptide (100 μM), which significantly increased whole-cell Ba2+ currents from 3.19 ± 0.34 pA/pF to 6.05 ± 0.43 pA/pF at 0 mV (n= 7, p<0.001, t-test). This effect of MANS peptide was abolished when cells were preincubated with wortmannin (20 μM). Immunocytochemistry and PLA studies demonstrated that MARCKS and PIP2 colocalise significantly at resting condition. However, upon stimulation with MANS peptide, this colocalization of MARCKS and PIP2 was reduced and CaV1.2 was shown to colocalise with PIP2 (n= 6, P<0.0001, t-test).
Conclusion:
These preliminary findings reveal the expression and interaction between MARCKS, CaV1.2 and PIP2 in cardiomyocytes, which likely controls cardiomyocytes calcium homeostasis. These molecular complexes may play an essential role in regulating cardiac function, and may be altered in cardiac dysfunction, thus providing a novel target for treating CVD.