Background: Intracellular Na ⁺ concentration ([Na ⁺ ] _i ) regulates Ca ²⁺ cycling, contractility, metabolism, and electrical stability of the heart. [Na ⁺ ] _i is elevated in heart failure, leading to arrhythmias and oxidative stress. We hypothesized that myocyte [Na ⁺ ] _i is also increased in type 2 diabetes (T2D) due to enhanced activity of the Na ⁺ –glucose cotransporter. Methods and Results: To test this hypothesis, we used myocardial tissue from humans with T2D and a rat model of late‐onset T2D ( HIP rat). Western blot analysis showed increased Na ⁺ –glucose cotransporter expression in failing hearts from T2D patients compared with nondiabetic persons (by 73±13%) and in HIP rat hearts versus wild‐type ( WT ) littermates (by 61±8%). [Na ⁺ ] _i was elevated in HIP rat myocytes both at rest (14.7±0.9 versus 11.4±0.7 mmol/L in WT ) and during electrical stimulation (17.3±0.8 versus 15.0±0.7 mmol/L); however, the Na ⁺ /K ⁺ ‐pump function was similar in HIP and WT cells, suggesting that higher [Na ⁺ ] _i is due to enhanced Na ⁺ entry in diabetic hearts. Indeed, Na ⁺ influx was significantly larger in myocytes from HIP versus WT rats (1.77±0.11 versus 1.29±0.06 mmol/L per minute). Na ⁺ –glucose cotransporter inhibition with phlorizin or glucose‐free solution greatly reduced Na ⁺ influx in HIP myocytes (to 1.20±0.16 mmol/L per minute), whereas it had no effect in WT cells. Phlorizin also significantly decreased glucose uptake in HIP myocytes (by 33±9%) but not in WT , indicating an increased reliance on the Na ⁺ –glucose cotransporter for glucose uptake in T2D hearts. Conclusions: Myocyte Na ⁺ –glucose cotransport is enhanced in T2D, which increases Na ⁺ influx and causes Na ⁺ overload. Higher [Na ⁺ ] _i may contribute to arrhythmogenesis and oxidative stress in diabetic hearts.

Rationale : Intracellular Ca ²⁺ concentration ([Ca ²⁺ ] _i ) is regulated and signals differently in various subcellular microdomains, which greatly enhances its second messenger versatility. In the heart, sarcoplasmic reticulum Ca ²⁺ release and signaling are controlled by local [Ca ²⁺ ] _i in the junctional cleft ([Ca ²⁺ ] _Cleft ), the small space between sarcolemma and junctional sarcoplasmic reticulum. However, methods to measure [Ca ²⁺ ] _Cleft directly are needed. Objective : To construct novel sensors that allow direct measurement of [Ca ²⁺ ] _Cleft . Methods and Results : We constructed cleft-targeted [Ca ²⁺ ] sensors by fusing Ca ²⁺ -sensor GCaMP2.2 and a new lower Ca ²⁺ -affinity variant GCaMP2.2Low to FKBP12.6, which binds with high affinity and selectivity to ryanodine receptors. The fluorescence pattern, affinity for ryanodine receptors, and competition by untagged FKBP12.6 demonstrated that FKBP12.6-tagged sensors are positioned to measure local [Ca ²⁺ ] _Cleft in adult rat myocytes. Using GCaMP2.2Low-FKBP12.6, we showed that [Ca ²⁺ ] _Cleft reaches higher levels with faster kinetics than global [Ca ²⁺ ] _i during excitation–contraction coupling. Diastolic sarcoplasmic reticulum Ca ²⁺ leak or sarcolemmal Ca ²⁺ entry may raise local [Ca ²⁺ ] _Cleft above bulk cytosolic [Ca ²⁺ ] _i ([Ca ²⁺ ] _Bulk ), an effect that may contribute to triggered arrhythmias and even transcriptional regulation. We measured this diastolic standing [Ca ²⁺ ] _Cleft –[Ca ²⁺ ] _Bulk gradient with GCaMP2.2-FKBP12.6 versus GCaMP2.2, using [Ca ²⁺ ] measured without gradients as a reference point. This diastolic difference ([Ca ²⁺ ] _Cleft =194 nmol/L versus [Ca ²⁺ ] _Bulk =100 nmol/L) is dictated mainly by the sarcoplasmic reticulum Ca ²⁺ leak rather than sarcolemmal Ca ²⁺ flux. Conclusions : We have developed junctional cleft-targeted sensors to measure [Ca ²⁺ ] _Cleft versus [Ca ²⁺ ] _Bulk and demonstrated dynamic differences during electric excitation and a standing diastolic [Ca ²⁺ ] _i gradient, which could influence local Ca ²⁺ -dependent signaling within the junctional cleft.

Loss-of-function mutations in the cytoskeletal protein ankyrin-B (AnkB) cause ventricular tachyarrhythmias in humans. Previously, we found that a larger fraction of the sarcoplasmic reticulum (SR) Ca ²⁺ leak occurs through Ca ²⁺ sparks in AnkB-deficient (AnkB ^+/− ) mice, which may contribute to arrhythmogenicity via Ca ²⁺ waves. Here, we investigated the mechanisms responsible for increased Ca ²⁺ spark frequency in AnkB ^+/− hearts. Using immunoblots and phospho-specific antibodies, we found that phosphorylation of ryanodine receptors (RyRs) by CaMKII is enhanced in AnkB ^+/− hearts. In contrast, the PKA-mediated RyR phosphorylation was comparable in AnkB ^+/− and wild-type (WT) mice. CaMKII inhibition greatly reduced Ca ²⁺ spark frequency in myocytes from AnkB ^+/− mice but had little effect in the WT. Global activities of the major phosphatases PP1 and PP2A were similar in AnkB ^+/− and WT hearts, while CaMKII autophosphorylation, a marker of CaMKII activation, was increased in AnkB ^+/− hearts. Thus, CaMKII-dependent RyR hyperphosphorylation in AnkB ^+/− hearts is caused by augmented CaMKII activity. Intriguingly, CaMKII activation is limited to the sarcolemma–SR junctions since non-junctional CaMKII targets (phospholamban, HDAC4) are not hyperphosphorylated in AnkB ^+/− myocytes. This local CaMKII activation may be the consequence of elevated [Ca ²⁺ ] in the junctional cleft caused by reduced Na ⁺ /Ca ²⁺ exchange activity. Indeed, using the RyR-targeted Ca ²⁺ sensor GCaMP2.2-FBKP12.6, we found that local junctional [Ca ²⁺ ] is significantly elevated in AnkB ^+/− myocytes. The increased incidence of pro-arrhythmogenic Ca ²⁺ sparks and waves in AnkB ^+/− hearts is due to enhanced CaMKII-mediated RyR phosphorylation, which is caused by higher junctional [Ca ²⁺ ] and consequent local CaMKII activation.

By controlling the function of various sarcolemmal and mitochondrial ion transporters, intracellular Na⁺ concentration ([Na⁺]_i) regulates Ca²⁺ cycling, electrical activity, the matching of energy supply and demand, and oxidative stress in cardiac myocytes. Thus, maintenance of myocyte Na⁺ homeostasis is vital for preserving the electrical and contractile activity of the heart. [Na⁺]_i is set by the balance between the passive Na⁺ entry through numerous pathways and the pumping of Na⁺ out of the cell by the Na⁺/K⁺-ATPase. This equilibrium is perturbed in heart failure, resulting in higher [Na⁺]_i. More recent studies have revealed that [Na⁺]_i is also increased in myocytes from diabetic hearts. Elevated [Na⁺]_i causes oxidative stress and augments the sarcoplasmic reticulum Ca²⁺ leak, thus amplifying the risk for arrhythmias and promoting heart dysfunction. This mini-review compares and contrasts the alterations in Na⁺ extrusion and/or Na⁺ uptake that underlie the [Na⁺]_i increase in heart failure and diabetes, with a particular emphasis on the emerging role of Na⁺ - glucose cotransporters in the diabetic heart.