The objective of the current study was to characterize calcium handling in developing human embryonic stem cell-derived cardiomyocytes (hESC-CMs). To this end, real-time polymerase chain reaction (PCR), immunocytochemistry, whole-cell voltage-clamp, and simultaneous patch-clamp/laser scanning confocal calcium imaging and surface membrane labeling with di-8-aminonaphthylethenylpridinium were used. Immunostaining studies in the hESC-CMs demonstrated the presence of the sarcoplasmic reticulum (SR) calcium release channels, ryanodine receptor-2, and inositol-1,4,5-trisphosphate (IP3) receptors. Store calcium function was manifested as action-potential-induced calcium transients. Time-to-target plots showed that these action-potential-initiated calcium transients traverse the width of the cell via a propagated wave of intracellular store calcium release. The hESC-CMs also exhibited local calcium events (“sparks”) that were localized to the surface membrane. The presence of caffeine-sensitive intracellular calcium stores was manifested following application of focal, temporally limited puffs of caffeine in three different age groups: early-stage (with the initiation of beating), intermediate-stage (10 days post-beating [dpb]), and late-stage (30–40 dpb) hESC-CMs. Calcium store load gradually increased during in vitro maturation. Similarly, ryanodine application decreased the amplitude of the spontaneous calcium transients. Interestingly, the expression and function of an IP3-releasable calcium pool was also demonstrated in the hESC-CMs in experiments using caged-IP3 photolysis and antagonist application (2 μM 2-Aminoethoxydiphenyl borate). In summary, our study establishes the presence of a functional SR calcium store in early-stage hESC-CMs and shows a unique pattern of calcium handling in these cells. This study also stresses the importance of the functional characterization of hESC-CMs both for developmental studies and for the development of future myocardial cell replacement strategies. Disclosure of potential conflicts of interest is found at the end of this article.

Calcium homeostasis is critical for cardiac myocyte function and must be tightly regulated. The guiding hypothesis of this study is that a carboxyl-terminal cleavage product of the cardiac L-type calcium channel (Ca _V 1.2) autoregulates expression. First, we confirmed that the Ca _V 1.2 C terminus (CCt) is cleaved in murine cardiac myocytes from mature and developing ventricle. Overexpression of full-length CCt caused a 34±8% decrease of Ca _V 1.2 promoter activity, and truncated CCt caused an 80±3% decrease of Ca _V 1.2 promoter (n=12). The full-length CCt distributes into cytosol and nucleus. A deletion mutant of CCt has a greater relative affinity for the nucleus than full-length CCt, and this is consistent with increased repression of Ca _V 1.2 promoter activity by truncated CCt. Chromatin immunoprecipitation analysis revealed that CCt interacts with the Ca _V 1.2 promoter in adult ventricular cardiac myocytes at promoter modules containing Nkx2.5/Mef2, C/EBp, and a cis regulatory module. The next hypothesis tested was that CCt contributes to transcriptional signaling associated with cellular hypertrophy. We explored whether fetal cardiac myocyte Ca _V 1.2 was regulated by serum in vitro. We tested atrial natriuretic factor promoter activity as a positive control and measured the serum response of Ca _V 1.2 promoter, protein, and L-type current ( I _Ca,L ) from fetal mouse ventricular myocytes. Serum increased atrial natriuretic factor promoter activity and cell size as expected. Serum withdrawal increased Ca _V 1.2 promoter activity, mRNA, and I _Ca,L . Moreover, serum withdrawal decreased the relative nuclear localization of CCt. A combination of promoter deletion mutant analyses, and the response of promoter mutants to serum withdrawal support the conclusion that CCt, a proteolytic fragment of Ca _V 1.2, autoregulates Ca _V 1.2 expression in cardiac myocytes. These data support the novel mechanism that a mobile segment of Ca _V 1.2 links Ca handling to nuclear signaling.

Background: Calmodulin (CaM) mutations have been identified recently in subjects with congenital long QT syndrome ( LQTS ) or catecholaminergic polymorphic ventricular tachycardia ( CPVT ), but the mechanisms responsible for these divergent arrhythmia‐susceptibility syndromes in this context are unknown. We tested the hypothesis that LQTS ‐associated CaM mutants disrupt Ca ²⁺ homeostasis in developing cardiomyocytes possibly by affecting either late Na current or Ca ²⁺ ‐dependent inactivation of L‐type Ca ²⁺ current. Methods and Results: We coexpressed CaM mutants with the human cardiac Na channel (Na _V 1.5) in tsA201 cells, and we used mammalian fetal ventricular cardiomyocytes to investigate LQTS ‐ and CPVT ‐associated CaM mutations ( LQTS ‐ and CPVT ‐CaM). LQTS ‐CaM mutants do not consistently affect L‐type Na current in heterologous cells or native cardiomyocytes, suggesting that the Na channel does not contribute to LQTS pathogenesis in the context of CaM mutations. LQTS ‐CaM mutants (D96V, D130G, F142L) impaired Ca ²⁺ ‐dependent inactivation, whereas the CPVT ‐CaM mutant N54I had no effect on Ca ²⁺ ‐dependent inactivation . LQTS ‐CaM mutants led to loss of Ca ²⁺ ‐transient entrainment with the rank order from greatest to least effect: CaM‐D130G~CaM‐D96V>>CaM‐F142L. This rank order follows measured Ca ²⁺ ‐CaM affinities for wild‐type and mutant CaM. Acute isoproterenol restored entrainment for CaM‐130G and CaM‐D96V but caused irreversible cytosolic Ca ²⁺ overload for cells expressing a CPVT ‐CaM mutant. Conclusions: CaM mutations associated with LQTS may not affect L‐type Na ⁺ current but may evoke defective Ca ²⁺ ‐dependent inactivation of L‐type Ca ²⁺ current.

Background: The regenerative capacity of the heart after myocardial infarction (MI) is limited. Our previous study showed that ectopic introduction of Cdk1/CyclinB1 and Cdk4/CyclinD1 complexes (4F) promotes cardiomyocyte proliferation in 15-20% of infected cardiomyocytes in vitro and in vivo and improves cardiac function after MI in mice. Methods: Here, using temporal single-cell RNAseq we aimed to identify the necessary reprogramming stages during the forced cardiomyocyte proliferation with 4F on a single cell basis. Also, using rat and pig models of ischemic heart failure, we aimed to start the first preclinical testing to introduce 4F gene therapy as a candidate for the treatment of ischemia-induced heart failure. Results: Temporal bulk and single-cell RNAseq and further biochemical validations of mature hiPS-CMs treated with either LacZ or 4F adenoviruses revealed full cell cycle reprogramming in 15% of the cardiomyocyte population at 48 h post-infection with 4F, which was mainly associated with sarcomere disassembly and metabolic reprogramming (n=3/timepoint/group). Transient overexpression of 4F, specifically in cardiomyocytes, was achieved using a polycistronic non-integrating lentivirus (NIL) encoding the 4F; each is driven by a TNNT2 promoter (TNNT2-4Fpolycistronic-NIL). TNNT2-4Fpolycistronic-NIL or control virus was injected intramyocardially one week after MI in rats (n=10/group) or pigs (n=6-7/group). Four weeks post-injection, TNNT2-4Fpolycistronic-NIL treated animals showed significant improvement in left ventricular ejection fraction and scar size compared with the control virus treated animals. At four months after treatment, rats that received TNNT2-4Fpolycistronic-NIL still showed a sustained improvement in cardiac function and no obvious development of cardiac arrhythmias or systemic tumorigenesis (n=10/group). Conclusions: This study provides mechanistic insights into the process of forced cardiomyocyte proliferation and advances the clinical feasibility of this approach by minimizing the oncogenic potential of the cell cycle factors thanks to the use of a novel transient and cardiomyocyte-specific viral construct.

Background: The small GTP ase Rad is a negative regulator of voltage‐dependent L‐type calcium channel current ( I _{C aL} ); however, the effects of Rad ablation on cardiomyocyte function are unknown. The objective of this study is to test the hypothesis that Rad‐depletion causes positive inotropic effects without inducing cardiac hypertrophy. Methods and Results: Ventricular myocytes from adult Rad ^−/− mice were isolated and evaluated by patch‐clamp recordings for I _Ca,L and action potentials, Ca ²⁺ transients, and sarcomere shortening. Maximum I _CaL is elevated in Rad ^−/− (maximal conductance 0.35±0.04 picoSiemens/picoFarad ( pS / pF ) wild‐type; 0.61±0.14 pS / pF Rad ^−/− ), decay kinetics are faster, and I _Ca,L activates at lower voltages (activation midpoint −7.2±0.6 wild‐type; −11.7±0.9 Rad ^−/− ) mimicking effects of β‐adrenergic receptor stimulation. Diastolic and twitch calcium are elevated in Rad ^−/− ( F _340/380 : 1.03 diastolic and 0.35 twitch for wild‐type; 1.47 diastolic and 0.736 twitch for Rad ^−/− ) and sarcomere shortening is enhanced (4.31% wild‐type; 14.13% Rad ^−/− ) at lower pacing frequencies. Consequentially, frequency‐dependence of Ca ²⁺ transients is less in Rad ^−/− , and the frequency dependence of relaxation is also blunted. In isolated working hearts, similar results were obtained; chiefly, + dP /dt was elevated at baseline and developed pressure was relatively nonresponsive to acute β‐adrenergic receptor stimulation. In single cells, at subphysiological frequencies, nonstimulated calmodulin‐dependent protein kinase–sensitive calcium release is observed. Remarkably, Rad ^−/− hearts did not show hypertrophic growth despite elevated levels of diastolic calcium. Conclusions: This study demonstrates that the depletion of Rad GTP ase is equivalent to sympathomimetic β‐adrenergic receptor, without stimulating cardiac hypertrophy. Thus, targeting Rad GTP ase is a novel potential therapeutic target for Ca ²⁺ ‐homeostasis–driven positive inotropic support of the heart.

Existing therapies to improve heart function target β-adrenergic receptor (β-AR) signaling and Ca²⁺ handling and often lead to adverse outcomes. This underscores an unmet need for positive inotropes that improve heart function without any adverse effects. The GTPase Ras associated with diabetes (RAD) regulates L-type Ca²⁺ channel (LTCC) current (I_Ca,L). Global RAD-knockout mice (gRAD^−/−) have elevated Ca²⁺ handling and increased cardiac hypertrophy, but RAD is expressed also in noncardiac tissues, suggesting the possibility that pathological remodeling is due also to noncardiac effects. Here, we engineered a myocardial-restricted inducible RAD-knockout mouse (RAD^Δ/Δ). Using an array of methods and techniques, including single-cell electrophysiological and calcium transient recordings, echocardiography, and radiotelemetry monitoring, we found that RAD deficiency results in a sustained increase of inotropy without structural or functional remodeling of the heart. I_Ca,L was significantly increased, with RAD loss conferring a β-AR–modulated phenotype on basal I_Ca,L. Cardiomyocytes from RAD^Δ/Δ hearts exhibited enhanced cytosolic Ca²⁺ handling, increased contractile function, elevated sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2a) expression, and faster lusitropy. These results argue that myocardial RAD ablation promotes a beneficial elevation in Ca²⁺ dynamics, which would obviate a need for increased β-AR signaling to improve cardiac function.