Dysregulated Rbfox2 produces aberrant splicing of CaV1.2 calcium channel in diabetes-induced cardiac hypertrophy

Abstract

Background: L-type Ca²⁺ channel Ca_V1.2 is essential for cardiomyocyte excitation, contraction and gene transcription in the heart, and abnormal functions of cardiac Ca_V1.2 channels are presented in diabetic cardiomyopathy. However, the underlying mechanisms are largely unclear. The functions of Ca_V1.2 channels are subtly modulated by splicing factor-mediated alternative splicing (AS), but whether and how Ca_V1.2 channels are alternatively spliced in diabetic heart remains unknown.

Methods: Diabetic rat models were established by using high-fat diet in combination with low dose streptozotocin. Cardiac function and morphology were assessed by echocardiography and HE staining, respectively. Isolated neonatal rat ventricular myocytes (NRVMs) were used as a cell-based model. Cardiac Ca_V1.2 channel functions were measured by whole-cell patch clamp, and intracellular Ca²⁺ concentration was monitored by using Fluo-4 AM.

Results: We find that diabetic rats develop diastolic dysfunction and cardiac hypertrophy accompanied by an increased Ca_V1.2 channel with alternative exon 9* (Ca_V1.2_E9*), but unchanged that with alternative exon 8/8a or exon 33. The splicing factor Rbfox2 expression is also increased in diabetic heart, presumably because of dominate-negative (DN) isoform. Unexpectedly, high glucose cannot induce the aberrant expressions of Ca_V1.2 exon 9* and Rbfox2. But glycated serum (GS), the mimic of advanced glycation end-products (AGEs), upregulates Ca_V1.2_E9* channels proportion and downregulates Rbfox2 expression in NRVMs. By whole-cell patch clamp, we find GS application hyperpolarizes the current-voltage curve and window currents of cardiac Ca_V1.2 channels. Moreover, GS treatment raises K⁺-triggered intracellular Ca²⁺ concentration ([Ca²⁺]_i), enlarges cell surface area of NRVMs and induces hypertrophic genes transcription. Consistently, siRNA-mediated knockdown of Rbfox2 in NRVMs upregulates Ca_V1.2_E9* channel, shifts Ca_V1.2 window currents to hyperpolarization, increases [Ca²⁺]_i and induces cardiomyocyte hypertrophy.

Conclusions: AGEs, not glucose, dysregulates Rbfox2 which thereby increases Ca_V1.2_E9* channels and hyperpolarizes channel window currents. These make the channels open at greater negative potentials and lead to increased [Ca²⁺]_i in cardiomyocytes, and finally induce cardiomyocyte hypertrophy in diabetes. Our work elucidates the underlying mechanisms for Ca_V1.2 channel regulation in diabetic heart, and targeting Rbfox2 to reset the aberrantly spliced Ca_V1.2 channel might be a promising therapeutic approach in diabetes-induced cardiac hypertrophy.

Keywords: Alternative splicing; CaV1.2 calcium channel; Cardiomyocyte hypertrophy; Diabetes.