Dysregulated Rbfox2 produces aberrant splicing of Ca1.2 calcium channel in diabetes-induced cardiac hypertrophy.

Background: L-type Ca channel Ca1.2 is essential for cardiomyocyte excitation, contraction and gene transcription in the heart, and abnormal functions of cardiac Ca1.2 channels are presented in diabetic cardiomyopathy. However, the underlying mechanisms are largely unclear. The functions of Ca1.2 channels are subtly modulated by splicing factor-mediated alternative splicing (AS), but whether and how Ca1.2 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 Ca1.2 channel functions were measured by whole-cell patch clamp, and intracellular Ca concentration was monitored by using Fluo-4 AM.

Results: We find that diabetic rats develop diastolic dysfunction and cardiac hypertrophy accompanied by an increased Ca1.2 channel with alternative exon 9* (Ca1.2), 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 Ca1.2 exon 9* and Rbfox2. But glycated serum (GS), the mimic of advanced glycation end-products (AGEs), upregulates Ca1.2 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 Ca1.2 channels. Moreover, GS treatment raises K-triggered intracellular Ca concentration ([Ca]), enlarges cell surface area of NRVMs and induces hypertrophic genes transcription. Consistently, siRNA-mediated knockdown of Rbfox2 in NRVMs upregulates Ca1.2 channel, shifts Ca1.2 window currents to hyperpolarization, increases [Ca] and induces cardiomyocyte hypertrophy.

Conclusions: AGEs, not glucose, dysregulates Rbfox2 which thereby increases Ca1.2 channels and hyperpolarizes channel window currents. These make the channels open at greater negative potentials and lead to increased [Ca] in cardiomyocytes, and finally induce cardiomyocyte hypertrophy in diabetes. Our work elucidates the underlying mechanisms for Ca1.2 channel regulation in diabetic heart, and targeting Rbfox2 to reset the aberrantly spliced Ca1.2 channel might be a promising therapeutic approach in diabetes-induced cardiac hypertrophy.