Diabetes increases oxidant stress and doubles the risk of dying after myocardial infarction, but the mechanisms underlying increased mortality are unknown. Mice with streptozotocin-induced diabetes developed profound heart rate slowing and doubled mortality compared with controls after myocardial infarction. Oxidized Ca 2+ /calmodulin-dependent protein kinase II (ox-CaMKII) was significantly increased in pacemaker tissues from diabetic patients compared with that in nondiabetic patients after myocardial infarction. Streptozotocin-treated mice had increased pacemaker cell ox-CaMKII and apoptosis, which were further enhanced by myocardial infarction. We developed a knockin mouse model of oxidation-resistant CaMKIIδ (MM-VV), the isoform associated with cardiovascular disease. Streptozotocin-treated MM-VV mice and WT mice infused with MitoTEMPO, a mitochondrial targeted antioxidant, expressed significantly less ox-CaMKII, exhibited increased pacemaker cell survival, maintained normal heart rates, and were resistant to diabetes-attributable mortality after myocardial infarction. Our findings suggest that activation of a mitochondrial/ox-CaMKII pathway contributes to increased sudden death in diabetic patients after myocardial infarction.

Background Arrhythmia is the major cause of death in patients with heart failure, for which β-adrenergic receptor (AR) blockers are a mainstay therapy. But the role of β-adrenergic signaling in electrophysiology and arrhythmias has never been studied in human ventricles. Methods and Results We used optical imaging of action potentials (AP) and [Ca2+]i transients (CaT) to compare the β1- and β2-adrenergic responses in left ventricular wedge preparations of human donor and failing hearts. β1-stimulation significantly increased conduction velocity (CV), shortened AP duration (APD), CaT duration (CaD) in donor but not failing hearts, due to desensitization of β1-AR in heart failure. In contrast, β2-stimulation increased CV in both donor and failing hearts but shortened APD only in failing hearts. β2-stimulation also affected transmural heterogeneity in APD but not in CaD. Both β1- and β2-stimulation augmented the vulnerability and frequency of ectopic activity and enhanced substrates for ventricular tachycardia in failing, but not donor, hearts. Both β1- and β2-stimulation enhanced Purkinje fiber automaticity, while only β2-stimulation promoted Ca-mediated premature ventricular contractions in heart failure. Conclusions During end-stage heart failure, β2-stimulation creates arrhythmogenic substrates via CV regulation and transmurally heterogeneous repolarization. β2-stimulation is, therefore, more arrhythmogenic than β1-stimulation. In particular, β2-stimulation increases the transmural difference between CaD and APD, which facilitates the formation of delayed afterdepolarizations.

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (V m ), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both V m and CaT simultaneously from the same location on the heart [1][2][3][4] . Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping 1,5,6 . In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of V m and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable. Video LinkThe 3. Prepare excitation-contraction uncoupler blebbistatin stock solution (Tocris Bioscience, St. Louis, MO, 2 mg/mL solution in DMSO) in advance and store the dissolved blebbistatin at 4°C.

Objective To look for previously unrecognized cardiac structural abnormalities and address the genetic cause for sudden unexplained nocturnal death syndrome (SUNDS). Methods and Results 148 SUNDS victims and 444 controls (matched 1:3 on gender, race, and age of death within 1 year) were collected from Sun Yat-sen University from January 1, 1998 to December 31, 2014 to search morphological changes. Additional 17 Brugada syndrome (BrS) patients collected from January 1, 2006 to December 31, 2014 served as a comparative disease cohort. The Target Captured Next Generation sequencing for 80 genes associated with arrhythmia/cardiomyopathy were performed in 44 SUNDS victims and 17 BrS patients to characterize the molecular spectrum. SUNDS had slight but statistically significantly increased heart weight and valve circumference compared to controls. 12/44 SUNDS victims (SCN5A, SCN1B, CACNB2, CACNA1C, AKAP9, KCNQ1, KCNH2, KCNJ5, GATA4, NUP155, ABCC9) and 6/17 BrS patients (SCN5A, CACNA1C, P>.05) carried rare variants in primary arrhythmia-susceptibility genes. Only 2/44 SUNDS cases compared to 5/17 BrS patients hosted a rare variant in the most common BrS causing gene, SCN5A (P=.01). Using the strict American College of Medical Genetics guideline-based definition, only 2/44 (KCNQ1) SUNDS and 3/17 (SCN5A) BrS patients hosted a “(likely) pathogenic” variant. The 14/44 SUNDS cases with cardiomyopathy-related variants had a subtle but significantly decreased circumference of cardiac valves, and tended to die on average 5–6 years younger compared to the remaining 30 cases (P=.02). Conclusions We present the first comprehensive autopsy evidence that SUNDS victims may have concealed cardiac morphological changes. SUNDS and BrS may result from different molecular pathological underpinnings. The distinct association between cardiomyopathy-related rare variants and SUNDS warrants further investigation.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) may provide an important bridge between animal models and intact human myocardium. Fulfilling this potential is hampered by their relative immaturity. hiPSC-CMs grown in monolayer culture lack a t-tubular system, have rudimentary intracellular calcium-handling systems, express predominantly embryonic sarcomeric protein isoforms, and preferentially use glucose as energy substrate. Culturing hiPSC-CM in a 3D environment and the addition of nutritional, pharmacologic and electromechanical stimuli have proven to be beneficial for maturation. We present an assessment of a model in which hiPSC-CMs and hiPSC-derived cardiac fibroblasts are co-cultured in a 3D fibrin matrix to form human engineered cardiac tissue constructs (hECT).The hECT respond to physiological stimuli, including stretch, frequency and β-adrenergic stimulation, develop a t-tubular system, and demonstrate calcium-handling and contractile kinetics that compare favorably with ventricular human myocardium. Transcript levels of genes involved in calcium-handling and contraction are increased. These markers of maturation become more robust over a short period of time in culture (6 weeks vs. 2 weeks in hECT). A comparison of the hECT molecular and performance variables with those of human cardiac tissue and other available engineered tissue platforms is provided to highlight strengths and weaknesses of these preparations. Important and noteworthy aspects of this human cardiac model system are its reliance on 'off-the-shelf' equipment, ability to provide detailed physiological performance data, and the ability to achieve a relatively mature cardiac physiology without additional nutritional, pharmacological and electromechanical stimuli that may elicit unintended effects on function.

Rationale Intracellular Ca2+ concentration ([Ca2+]i) is regulated and signals differently in various subcellular microdomains, which greatly enhances its second messenger versatility. In the heart, sarcoplasmic reticulum (SR) Ca2+ release and signaling is controlled by local [Ca2+]i in the junctional cleft ([Ca2+]Cleft), the small space between sarcolemma and junctional SR. However, methods to directly measure [Ca2+]Cleft are needed. Objective To construct novel sensors that allow direct measurement of [Ca2+]Cleft. Methods and Results We constructed cleft-targeted [Ca2+] sensors by fusing Ca2+-sensor GCaMP2.2 and a new lower Ca2+-affinity variant GCaMP2.2Low to FKBP12.6, which binds with high affinity and selectivity to ryanodine receptors (RyRs). The fluorescence pattern, affinity for RyRs and competition by un-tagged FKBP12.6 demonstrated that FKBP12.6-tagged sensors are positioned to measure local [Ca2+]Cleft in adult rat myocytes. Using GCaMP2.2Low-FKBP12.6, we showed that [Ca2+]Cleft reaches higher levels with faster kinetics than global [Ca2+]i during excitation-contraction coupling. Diastolic SR Ca2+ leak or sarcolemmal Ca2+ entry may raise local [Ca2+]Cleft above bulk cytosolic [Ca2+]i ([Ca2+]Bulk), an effect that may contribute to triggered arrhythmias and even transcriptional regulation. We measured this diastolic standing [Ca2+]Cleft–[Ca2+]Bulk gradient using GCaMP2.2-FKBP12.6 vs. GCaMP2.2, using [Ca2+] measured without gradients as a reference point. This diastolic difference ([Ca2+]Cleft=194 nmol/L vs. [Ca2+]Bulk=100 nmol/L) is dictated mainly by the SR Ca2+ leak, rather than sarcolemmal Ca2+ flux. Conclusions We have developed junctional cleft targeted sensors to measure [Ca2+]Cleft vs. [Ca2+]Bulk, and demonstrated dynamic differences during electrical excitation and a standing diastolic [Ca2+]i gradient which could influence local Ca2+-dependent signaling within the junctional cleft.

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Sinoatrial node is responsible for the origin of the wave of excitation, which spreads throughout the heart and orchestrates cardiac contraction via calcium-mediated excitation-contraction coupling. P-wave represents the spread of excitation in the atria. It is well known that the autonomic nervous system controls the heart rate by dynamically altering both cellular ionic fluxes and the anatomic location of the leading pacemaker. In this study we used isolated rabbit right atria and mathematical model of the pacemaker region of the rabbit heart. Application of isoproterenol resulted in doze-dependent acceleration of the heart rate and superior shift of the leading pacemaker. In the mathematical model such behavior could be reproduced by a gradient of expression in β1-adrenergic receptors along the superior-inferior axis. Application of acetylcholine resulted in preferentially inferior shift of pacemaker and slowing of the heart rate. The mathematical model reproduced this behavior with imposing a gradient of expression of acetylcholine-sensitive potassium channel. We conclude that anatomical shift of the leading pacemaker in the rabbit heart could be achieved through gradient of expression of β1-adrenergic receptors and IK,ACh.