Rationale: Pathological cardiac myocyte hypertrophy is thought to be induced by the persistent increases in intracellular Ca ²⁺ needed to maintain cardiac function when systolic wall stress is increased. Hypertrophic Ca ²⁺ binds to calmodulin (CaM) and activates the phosphatase calcineurin (Cn) and CaM kinase (CaMK)II. Cn dephosphorylates cytoplasmic NFAT (nuclear factor of activated T cells), inducing its translocation to the nucleus where it activates antiapoptotic and hypertrophic target genes. Cytoplasmic CaMKII regulates Ca ²⁺ handling proteins but whether or not it is directly involved in hypertrophic and survival signaling is not known. Objective: This study explored the hypothesis that cytoplasmic CaMKII reduces NFAT nuclear translocation by inhibiting the phosphatase activity of Cn. Methods and Results: Green fluorescent protein–tagged NFATc3 was used to determine the cellular location of NFAT in cultured neonatal rat ventricular myocytes (NRVMs) and adult feline ventricular myocytes. Constitutively active (CaMKII-CA) or dominant negative (CaMKII-DN) mutants of cytoplasmic targeted CaMKII _δc were used to activate and inhibit cytoplasmic CaMKII activity. In NRVM CaMKII-DN (48.5±3%, P <0.01 versus control) increased, whereas CaMKII-CA decreased (5.9±1%, P <0.01 versus control) NFAT nuclear translocation (Control: 12.3±1%). Cn inhibitors were used to show that these effects were caused by modulation of Cn activity. Increasing Ca ²⁺ increased Cn-dependent NFAT translocation (to 71.7±7%, P <0.01) and CaMKII-CA reduced this effect (to 17.6±4%). CaMKII-CA increased TUNEL and caspase-3 activity ( P <0.05). CaMKII directly phosphorylated Cn at Ser197 in CaMKII-CA infected NRVMs and in hypertrophied feline hearts. Conclusion: These data show that activation of cytoplasmic CaMKII inhibits NFAT nuclear translocation by phosphorylation and subsequent inhibition of Cn.

Depressed contractility of failing myocytes involves a decreased rate of rise of the Ca ²⁺ transient. Synchronization of Ca ²⁺ release from the junctional sarcoplasmic reticulum (SR) is responsible for the rapid rise of the normal Ca ²⁺ transient. This study examined the idea that spatially and temporally dyssynchronous SR Ca ²⁺ release slows the rise of the cytosolic Ca ²⁺ transient in failing feline myocytes. Left ventricular hypertrophy (LVH) with and without heart failure (HF) was induced in felines by constricting the ascending aorta. Ca ²⁺ transients were measured in ventricular myocytes using confocal line scan imaging. Ca ²⁺ transients were induced by field stimulation, square wave voltage steps, or action potential (AP) voltage clamp. SR Ca ²⁺ release was significantly less well spatially and temporally synchronized in field-stimulated HF versus control or LVH myocytes. Surprisingly, depolarization of HF cells to potentials where Ca ²⁺ currents ( I _Ca ) were maximal resynchronized SR Ca ²⁺ release. Correspondingly, decreases in the amplitude of I _Ca desynchronized SR Ca ²⁺ release in control, LVH, and HF myocytes to the same extent. HF myocytes had significant loss of phase 1 AP repolarization and smaller I _Ca density, which should both reduce Ca ²⁺ influx. When normal myocytes were voltage clamped with HF AP profiles SR Ca ²⁺ release was desynchronized. SR Ca ²⁺ release becomes dyssynchronized in failing feline ventricular myocytes because of reductions in Ca ²⁺ influx induced in part by alterations in early repolarization of the AP. Therefore, therapies that restore normal early repolarization should improve the contractility of the failing heart.

T-type Ca ²⁺ channels (TTCCs) are expressed in the developing heart, are not present in the adult ventricle, and are reexpressed in cardiac diseases involving cardiac dysfunction and premature, arrhythmogenic death. The goal of this study was to determine the functional role of increased Ca ²⁺ influx through reexpressed TTCCs in the adult heart. A mouse line with cardiac-specific, conditional expression of the α1G-TTCC was used to increase Ca ²⁺ influx through TTCCs. α1G hearts had mild increases in contractility but no cardiac histopathology or premature death. This contrasts with the pathological phenotype of a previously studied mouse with increased Ca ²⁺ influx through the L-type Ca ²⁺ channel (LTCC) secondary to overexpression of its β2a subunit. Although α1G and β2a myocytes had similar increases in Ca ²⁺ influx, α1G myocytes had smaller increases in contraction magnitude, and, unlike β2a myocytes, there were no increases in sarcoplasmic reticulum Ca ²⁺ loading. Ca ²⁺ influx through TTCCs also did not induce normal sarcoplasmic reticulum Ca ²⁺ release. α1G myocytes had changes in LTCC, SERCA2a, and phospholamban abundance, which appear to be adaptations that help maintain Ca ²⁺ homeostasis. Immunostaining suggested that the majority of α1G-TTCCs were on the surface membrane. Osmotic shock, which selectively eliminates T-tubules, induced a greater reduction in L- versus TTCC currents. These studies suggest that T- and LTCCs are in different portions of the sarcolemma (surface membrane versus T-tubules) and that Ca ²⁺ influx through these channels induce different effects on myocyte contractility and lead to distinct cardiac phenotypes.

Cardiac diseases such as myocardial infarction (MI) can lead to adverse remodeling and impaired contractility of the heart due to widespread cardiomyocyte death in the damaged area. Current therapies focus on improving heart contractility and minimizing fibrosis with modest cardiac regeneration, but MI patients can still progress to heart failure (HF). There is a dire need for clinical therapies that can replace the lost myocardium, specifically by the induction of new myocyte formation from pre-existing cardiomyocytes. Many studies have shown terminally differentiated myocytes can re-enter the cell cycle and divide through manipulations of the cardiomyocyte cell cycle, signaling pathways, endogenous genes, and environmental factors. However, these approaches result in minimal myocyte renewal or cardiomegaly due to hyperactivation of cardiomyocyte proliferation. Finding the optimal treatment that will replenish cardiomyocyte numbers without causing tumorigenesis is a major challenge in the field. Another controversy is the inability to clearly define cardiomyocyte division versus myocyte DNA synthesis due to limited methods. In this review, we discuss several studies that induced cardiomyocyte cell cycle re-entry after cardiac injury, highlight whether cardiomyocytes completed cytokinesis, and address both limitations and methodological advances made to identify new myocyte formation.

The utility of bone marrow cells (BMCs) to regenerate cardiac myocytes is controversial. The present study examined the capacity of different types of BMCs to generate functional cardiac myocytes. Isolated c‐kit⁺ BMCs (BMSCs), c‐kit⁺ and crude BMCs from the adult feline femur were membrane stained with PKH26 dye or infected with a control enhanced green fluorescence protein transcript (EGFP)‐adenovirus prior to co‐culture upon neonatal rat ventricular myocytes (NRVM). Co‐cultured cells were immuno‐stained for c‐kit, α‐tropomyosin, α‐actinin, connexin 43 (C×43) and Ki67 and analyzed with confocal microscopy. Electrophysiology of BMSC derived myocytes were compared to NRVMs within the same culture dish. Gap junction function was analyzed by fluorescence recovery after photo‐bleaching (FRAP). BMCs proliferated and differentiated into cardiac myocytes during the first 48 hours of co‐culturing. These newly formed cardiac myocytes were able to contract spontaneously or synchronously with neighboring NRVMs. The myogenic rate of c‐kit⁺ BMSCs was significantly greater than c‐kit⁺ and crude BMCs (41.2 ± 2.1, 6.1 ± 1.2, and 17.1 ± 1.5%, respectively). The newly formed cardiac myocytes exhibited an immature electrophysiological phenotype until they became electrically coupled to NRVMs through functional gap junctions. BMSCs did not become functional myocytes in the absence of NRVMs. In conclusion, c‐kit⁺ BMSCs have the ability to transdifferentiate into functional cardiac myocytes.