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

BackgroundL-type Ca2+ channel Ca(V)1.2 is essential for cardiomyocyte excitation, contraction and gene transcription in the heart, and abnormal functions of cardiac Ca(V)1.2 channels are presented in diabetic cardiomyopathy. However, the underlying mechanisms are largely unclear. The functions of Ca(V)1.2 channels are subtly modulated by splicing factor-mediated alternative splicing (AS), but whether and how Ca(V)1.2 channels are alternatively spliced in diabetic heart remains unknown.MethodsDiabetic 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(V)1.2 channel functions were measured by whole-cell patch clamp, and intracellular Ca2+ concentration was monitored by using Fluo-4 AM.ResultsWe find that diabetic rats develop diastolic dysfunction and cardiac hypertrophy accompanied by an increased Ca(V)1.2 channel with alternative exon 9* (Ca(V)1.2(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(V)1.2 exon 9* and Rbfox2. But glycated serum (GS), the mimic of advanced glycation end-products (AGEs), upregulates Ca(V)1.2(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(V)1.2 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(V)1.2(E9*) channel, shifts Ca(V)1.2 window currents to hyperpolarization, increases [Ca2+](i) and induces cardiomyocyte hypertrophy.ConclusionsAGEs, not glucose, dysregulates Rbfox2 which thereby increases Ca(V)1.2(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(V)1.2 channel regulation in diabetic heart, and targeting Rbfox2 to reset the aberrantly spliced Ca(V)1.2 channel might be a promising therapeutic approach in diabetes-induced cardiac hypertrophy.

Automated summary

Diabetic heart exhibits alternative splicing of Ca(V)1.2 channels, especially the Ca(V)1.2(E9*) channel, which is responsible for altered calcium concentration and cardiomyocyte hypertrophy. Glycated serum regulates the expression of Rbfox2, thereby affecting the function of these channels.