Stefan Neef

Generation of Functional Murine Cardiac Myocytes From Induced Pluripotent Stem Cells

Background— The recent breakthrough in the generation of induced pluripotent stem (iPS) cells, which are almost indistinguishable from embryonic stem (ES) cells, facilitates the generation of murine disease– and human patient–specific stem cell lines. The aim of this study was to characterize the cardiac differentiation potential of a murine iPS cell clone in comparison to a well-established murine ES cell line. Methods and Results— With the use of a standard embryoid body–based differentiation protocol for ES cells, iPS cells as well as ES cells were differentiated for 24 days. Although the analyzed iPS cell clone showed a delayed and less efficient formation of beating embryoid bodies compared with the ES cell line, the differentiation resulted in an average of 55% of spontaneously contracting iPS cell embryoid bodies. Analyses on molecular, structural, and functional levels demonstrated that iPS cell–derived cardiomyocytes show typical features of ES cell–derived cardiomyocytes. Reverse transcription polymerase chain reaction analyses demonstrated expression of marker genes typical for mesoderm, cardiac mesoderm, and cardiomyocytes including Brachyury, mesoderm posterior factor 1 (Mesp1), friend of GATA2 (FOG-2), GATA-binding protein 4 (GATA4), NK2 transcription factor related, locus 5 (Nkx2.5), T-box 5 (Tbx5), T-box 20 (Tbx20), atrial natriuretic factor (ANF), myosin light chain 2 atrial transcripts (MLC2a), myosin light chain 2 ventricular transcripts (MLC2v), &agr;-myosin heavy chain (&agr;-MHC), and cardiac troponin T in differentiation cultures of iPS cells. Immunocytology confirmed expression of cardiomyocyte-typical proteins including sarcomeric &agr;-actinin, titin, cardiac troponin T, MLC2v, and connexin 43. iPS cell cardiomyocytes displayed spontaneous rhythmic intracellular Ca2+ fluctuations with amplitudes of Ca2+ transients comparable to ES cell cardiomyocytes. Simultaneous Ca2+ release within clusters of iPS cell–derived cardiomyocytes indicated functional coupling of the cells. Electrophysiological studies with multielectrode arrays demonstrated functionality and presence of the &bgr;-adrenergic and muscarinic signaling cascade in these cells. Conclusions— iPS cells differentiate into functional cardiomyocytes. In contrast to ES cells, iPS cells allow derivation of autologous functional cardiomyocytes for cellular cardiomyoplasty and myocardial tissue engineering.

The δ isoform of CaM kinase II is required for pathological cardiac hypertrophy and remodeling after pressure overload

Acute and chronic injuries to the heart result in perturbation of intracellular calcium signaling, which leads to pathological cardiac hypertrophy and remodeling. Calcium/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the transduction of calcium signals in the heart, but the specific isoforms of CaMKII that mediate pathological cardiac signaling have not been fully defined. To investigate the potential involvement in heart disease of CaMKIIδ, the major CaMKII isoform expressed in the heart, we generated CaMKIIδ-null mice. These mice are viable and display no overt abnormalities in cardiac structure or function in the absence of stress. However, pathological cardiac hypertrophy and remodeling are attenuated in response to pressure overload in these animals. Cardiac extracts from CaMKIIδ-null mice showed diminished kinase activity toward histone deacetylase 4 (HDAC4), a substrate of stress-responsive protein kinases and suppressor of stress-dependent cardiac remodeling. In contrast, phosphorylation of the closely related HDAC5 was unaffected in hearts of CaMKIIδ-null mice, underscoring the specificity of the CaMKIIδ signaling pathway for HDAC4 phosphorylation. We conclude that CaMKIIδ functions as an important transducer of stress stimuli involved in pathological cardiac remodeling in vivo, which is mediated, at least in part, by the phosphorylation of HDAC4. These findings point to CaMKIIδ as a potential therapeutic target for the maintenance of cardiac function in the setting of pressure overload.

CaMKII-Dependent Diastolic SR Ca2+ Leak and Elevated Diastolic Ca2+ Levels in Right Atrial Myocardium of Patients With Atrial Fibrillation

Rationale: Although research suggests that diastolic Ca2+ levels might be increased in atrial fibrillation (AF), this hypothesis has never been tested. Diastolic Ca2+ leak from the sarcoplasmic reticulum (SR) might increase diastolic Ca2+ levels and play a role in triggering or maintaining AF by transient inward currents through Na+/Ca2+ exchange. In ventricular myocardium, ryanodine receptor type 2 (RyR2) phosphorylation by Ca2+/calmodulin-dependent protein kinase (CaMK)II is emerging as an important mechanism for SR Ca2+ leak. Objective: We tested the hypothesis that CaMKII-dependent diastolic SR Ca2+ leak and elevated diastolic Ca2+ levels occurs in atrial myocardium of patients with AF. Methods and Results: We used isolated human right atrial myocytes from patients with AF versus sinus rhythm and found CaMKII expression to be increased by 40±14% (P<0.05), as well as CaMKII phosphorylation by 33±12% (P<0.05). This was accompanied by a significantly increased RyR2 phosphorylation at the CaMKII site (Ser2814) by 110±53%. Furthermore, cytosolic Ca2+ levels were elevated during diastole (229±20 versus 164±8 nmol/L, P<0.05). Most likely, this resulted from an increased SR Ca2+ leak in AF (P<0.05), which was not attributable to higher SR Ca2+ load. Tetracaine experiments confirmed that SR Ca2+ leak through RyR2 leads to the elevated diastolic Ca2+ level. CaMKII inhibition normalized SR Ca2+ leak and cytosolic Ca2+ levels without changes in L-type Ca2+ current. Conclusion: Increased CaMKII-dependent phosphorylation of RyR2 leads to increased SR Ca2+ leak in human AF, causing elevated cytosolic Ca2+ levels, thereby providing a potential arrhythmogenic substrate that could trigger or maintain AF.

Inhibition of Elevated Ca2+/Calmodulin-Dependent Protein Kinase II Improves Contractility in Human Failing Myocardium

Rationale: Heart failure (HF) is known to be associated with increased Ca2+/calmodulin-dependent protein kinase (CaMK)II expression and activity. There is still controversial discussion about the functional role of CaMKII in HF. Moreover, CaMKII inhibition has never been investigated in human myocardium. Objective: We sought to investigate detailed CaMKII&dgr; expression in end-stage failing human hearts (dilated and ischemic cardiomyopathy) and the functional effects of CaMKII inhibition on contractility. Methods and Results: Expression analysis revealed that CaMKII&dgr;, both cytosolic &dgr;C and nuclear &dgr;B splice variants, were significantly increased in both right and left ventricles from patients with dilated or ischemic cardiomyopathy versus nonfailing. Experiments with isometrically twitching trabeculae revealed significantly improved force frequency relationships in the presence of CaMKII inhibitors (KN-93 and AIP). Increased postrest twitches after CaMKII inhibition indicated an improved sarcoplasmic reticulum (SR) Ca2+ loading. This was confirmed in isolated myocytes by a reduced SR Ca2+ spark frequency and hence SR Ca2+ leak, resulting in increased SR Ca2+ load when inhibiting CaMKII. Ryanodine receptor type 2 phosphorylation at Ser2815, which is known to be phosphorylated by CaMKII thereby contributing to SR Ca2+ leak, was found to be markedly reduced in KN-93–treated trabeculae. Interestingly, CaMKII inhibition did not influence contractility in nonfailing sheep trabeculae. Conclusions: The present study shows for the first time that CaMKII inhibition acutely improves contractility in human HF where CaMKII&dgr; expression is increased. The mechanism proposed consists of a reduced SR Ca2+ leak and consequently increased SR Ca2+ load. Thus, CaMKII inhibition appears to be a possible therapeutic option for patients with HF and merits further investigation.

Calcium/Calmodulin-Dependent Protein Kinase II Contributes to Cardiac Arrhythmogenesis in Heart Failure

Background —Transgenic CaMKIIδC (TG) mice have heart failure and isoproterenol (ISO)-inducible arrhythmias. We hypothesized that CaMKII contributes to arrhythmias and to underlying cellular events and that inhibition of CaMKII reduces cardiac arrhythmogenesis in vitro and in vivo. Methods and Results —Under baseline conditions, isolated cardiac myocytes from TG mice revealed an increased incidence of early afterdepolarizations (ADs) as compared to wild-type (WT) myocytes (P<0.05). CaMKII-inhibition (AIP) completely abolished these ADs in TG cells ( P <0.05). Elevating intracellular Ca stores using ISO (10-8 M) induced a larger amount of delayed ADs and spontaneous action potentials in TG as compared to WT ( P <0.05). This seems to be due to an increased sarcoplasmic reticulum (SR) Ca leak since diastolic [Ca] rose clearly upon ISO in TG but not in WT cells (+20±5% vs. +3±4% at 10-6 M ISO, P <0.05). In parallel, SR Ca leak assessed by spontaneous SR Ca release events showed an increased Ca spark frequency (3.9±0.5 vs. 2.0±0.4 sparks per 100 μm-1*s-1, P <0.05). However, CaMKII-inhibition (either pharmacologically using KN-93 or genetically using an isoform-specific CaMKIIδ-knockout mouse model) significantly reduced SR Ca spark frequency although this rather increased SR Ca content. In parallel, ISO increased the incidence of early (54% vs. 4%, P <0.05) and late (86% vs. 43%, P <0.05) non-stimulated events (NSEs) in TG vs. WT myocytes but CaMKII-inhibition (KN-93 and KO) reduced these proarrhythmogenic events ( P <0.05). In addition, CaMKII-inhibition in TG mice (KN-93) clearly reduced ISO-induced arrhythmias in vivo ( P <0.05). Conclusions —We conclude that CaMKII contributes to cardiac arrhythmogenesis in TG CaMKIIδC mice having heart failure and suggest the increased SR Ca leak as an important mechanism. Moreover, CaMKII-inhibition reduces cardiac arrhythmias in vitro and in vivo and may therefore indicate a potential role for future antiarrhythmic therapies warranting further studies.Background—Transgenic (TG) Ca/calmodulin-dependent protein kinase II (CaMKII)&dgr;C mice have heart failure and isoproterenol (ISO)-inducible arrhythmias. We hypothesized that CaMKII contributes to arrhythmias and underlying cellular events and that inhibition of CaMKII reduces cardiac arrhythmogenesis in vitro and in vivo. Methods and Results—Under baseline conditions, isolated cardiac myocytes from TG mice showed an increased incidence of early afterdepolarizations compared with wild-type myocytes (P<0.05). CaMKII inhibition (AIP) completely abolished these afterdepolarizations in TG cells (P<0.05). Increasing intracellular Ca stores using ISO (10−8 M) induced a larger amount of delayed afterdepolarizations and spontaneous action potentials in TG compared with wild-type cells (P<0.05). This seems to be due to an increased sarcoplasmic reticulum (SR) Ca leak because diastolic [Ca]i rose clearly on ISO in TG but not in wild-type cells (+20±5% versus +3±4% at 10−6 M ISO, P<0.05). In parallel, SR Ca leak assessed by spontaneous SR Ca release events showed an increased Ca spark frequency (3.9±0.5 versus 2.0±0.4 sparks per 100 &mgr;m−1·s−1, P<0.05). However, CaMKII inhibition (either pharmacologically using KN-93 or genetically using an isoform-specific CaMKII&dgr;-knockout mouse model) significantly reduced SR Ca spark frequency, although this rather increased SR Ca content. In parallel, ISO increased the incidence of early (54% versus 4%, P<0.05) and late (86% versus 43%, P<0.05) nonstimulated events in TG versus wild-type myocytes, but CaMKII inhibition (KN-93 and KO) reduced these proarrhythmogenic events (P<0.05). In addition, CaMKII inhibition in TG mice (KN-93) clearly reduced ISO-induced arrhythmias in vivo (P<0.05). Conclusions—We conclude that CaMKII contributes to cardiac arrhythmogenesis in TG CaMKII&dgr;C mice having heart failure and suggest the increased SR Ca leak as an important mechanism. Moreover, CaMKII inhibition reduces cardiac arrhythmias in vitro and in vivo and may therefore indicate a potential role for future antiarrhythmic therapies warranting further studies.

Oxidized Ca 2+ /Calmodulin-Dependent Protein Kinase II Triggers Atrial Fibrillation

Background —Atrial fibrillation is a growing public health problem without adequate therapies. Angiotensin II (Ang II) and reactive oxygen species (ROS) are validated risk factors for atrial fibrillation (AF) in patients, but the molecular pathway(s) connecting ROS and AF is unknown. The Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) has recently emerged as a ROS activated proarrhythmic signal, so we hypothesized that oxidized CaMKIIδ(ox-CaMKII) could contribute to AF. Methods and Results —We found ox-CaMKII was increased in atria from AF patients compared to patients in sinus rhythm and from mice infused with Ang II compared with saline. Ang II treated mice had increased susceptibility to AF compared to saline treated WT mice, establishing Ang II as a risk factor for AF in mice. Knock in mice lacking critical oxidation sites in CaMKIIδ (MM-VV) and mice with myocardial-restricted transgenic over-expression of methionine sulfoxide reductase A (MsrA TG), an enzyme that reduces ox-CaMKII, were resistant to AF induction after Ang II infusion. Conclusions —Our studies suggest that CaMKII is a molecular signal that couples increased ROS with AF and that therapeutic strategies to decrease ox-CaMKII may prevent or reduce AF.Background— Atrial fibrillation (AF) is a growing public health problem without adequate therapies. Angiotensin II and reactive oxygen species are validated risk factors for AF in patients, but the molecular pathways connecting reactive oxygen species and AF are unknown. The Ca2+/calmodulin-dependent protein kinase II (CaMKII) has recently emerged as a reactive oxygen species–activated proarrhythmic signal, so we hypothesized that oxidized CaMKII&dgr; could contribute to AF. Methods and Results— We found that oxidized CaMKII was increased in atria from AF patients compared with patients in sinus rhythm and from mice infused with angiotensin II compared with mice infused with saline. Angiotensin II–treated mice had increased susceptibility to AF compared with saline-treated wild-type mice, establishing angiotensin II as a risk factor for AF in mice. Knock-in mice lacking critical oxidation sites in CaMKII&dgr; (MM-VV) and mice with myocardium-restricted transgenic overexpression of methionine sulfoxide reductase A, an enzyme that reduces oxidized CaMKII, were resistant to AF induction after angiotensin II infusion. Conclusions— Our studies suggest that CaMKII is a molecular signal that couples increased reactive oxygen species with AF and that therapeutic strategies to decrease oxidized CaMKII may prevent or reduce AF.

CaMKII Contributes to Cardiac Arrhythmogenesis in Heart Failure

Background —Transgenic CaMKIIδC (TG) mice have heart failure and isoproterenol (ISO)-inducible arrhythmias. We hypothesized that CaMKII contributes to arrhythmias and to underlying cellular events and that inhibition of CaMKII reduces cardiac arrhythmogenesis in vitro and in vivo. Methods and Results —Under baseline conditions, isolated cardiac myocytes from TG mice revealed an increased incidence of early afterdepolarizations (ADs) as compared to wild-type (WT) myocytes (P<0.05). CaMKII-inhibition (AIP) completely abolished these ADs in TG cells ( P <0.05). Elevating intracellular Ca stores using ISO (10-8 M) induced a larger amount of delayed ADs and spontaneous action potentials in TG as compared to WT ( P <0.05). This seems to be due to an increased sarcoplasmic reticulum (SR) Ca leak since diastolic [Ca] rose clearly upon ISO in TG but not in WT cells (+20±5% vs. +3±4% at 10-6 M ISO, P <0.05). In parallel, SR Ca leak assessed by spontaneous SR Ca release events showed an increased Ca spark frequency (3.9±0.5 vs. 2.0±0.4 sparks per 100 μm-1*s-1, P <0.05). However, CaMKII-inhibition (either pharmacologically using KN-93 or genetically using an isoform-specific CaMKIIδ-knockout mouse model) significantly reduced SR Ca spark frequency although this rather increased SR Ca content. In parallel, ISO increased the incidence of early (54% vs. 4%, P <0.05) and late (86% vs. 43%, P <0.05) non-stimulated events (NSEs) in TG vs. WT myocytes but CaMKII-inhibition (KN-93 and KO) reduced these proarrhythmogenic events ( P <0.05). In addition, CaMKII-inhibition in TG mice (KN-93) clearly reduced ISO-induced arrhythmias in vivo ( P <0.05). Conclusions —We conclude that CaMKII contributes to cardiac arrhythmogenesis in TG CaMKIIδC mice having heart failure and suggest the increased SR Ca leak as an important mechanism. Moreover, CaMKII-inhibition reduces cardiac arrhythmias in vitro and in vivo and may therefore indicate a potential role for future antiarrhythmic therapies warranting further studies.Background—Transgenic (TG) Ca/calmodulin-dependent protein kinase II (CaMKII)&dgr;C mice have heart failure and isoproterenol (ISO)-inducible arrhythmias. We hypothesized that CaMKII contributes to arrhythmias and underlying cellular events and that inhibition of CaMKII reduces cardiac arrhythmogenesis in vitro and in vivo. Methods and Results—Under baseline conditions, isolated cardiac myocytes from TG mice showed an increased incidence of early afterdepolarizations compared with wild-type myocytes (P<0.05). CaMKII inhibition (AIP) completely abolished these afterdepolarizations in TG cells (P<0.05). Increasing intracellular Ca stores using ISO (10−8 M) induced a larger amount of delayed afterdepolarizations and spontaneous action potentials in TG compared with wild-type cells (P<0.05). This seems to be due to an increased sarcoplasmic reticulum (SR) Ca leak because diastolic [Ca]i rose clearly on ISO in TG but not in wild-type cells (+20±5% versus +3±4% at 10−6 M ISO, P<0.05). In parallel, SR Ca leak assessed by spontaneous SR Ca release events showed an increased Ca spark frequency (3.9±0.5 versus 2.0±0.4 sparks per 100 &mgr;m−1·s−1, P<0.05). However, CaMKII inhibition (either pharmacologically using KN-93 or genetically using an isoform-specific CaMKII&dgr;-knockout mouse model) significantly reduced SR Ca spark frequency, although this rather increased SR Ca content. In parallel, ISO increased the incidence of early (54% versus 4%, P<0.05) and late (86% versus 43%, P<0.05) nonstimulated events in TG versus wild-type myocytes, but CaMKII inhibition (KN-93 and KO) reduced these proarrhythmogenic events (P<0.05). In addition, CaMKII inhibition in TG mice (KN-93) clearly reduced ISO-induced arrhythmias in vivo (P<0.05). Conclusions—We conclude that CaMKII contributes to cardiac arrhythmogenesis in TG CaMKII&dgr;C mice having heart failure and suggest the increased SR Ca leak as an important mechanism. Moreover, CaMKII inhibition reduces cardiac arrhythmias in vitro and in vivo and may therefore indicate a potential role for future antiarrhythmic therapies warranting further studies.

Oxidized CaMKII Triggers Atrial Fibrillation

Background —Atrial fibrillation is a growing public health problem without adequate therapies. Angiotensin II (Ang II) and reactive oxygen species (ROS) are validated risk factors for atrial fibrillation (AF) in patients, but the molecular pathway(s) connecting ROS and AF is unknown. The Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) has recently emerged as a ROS activated proarrhythmic signal, so we hypothesized that oxidized CaMKIIδ(ox-CaMKII) could contribute to AF. Methods and Results —We found ox-CaMKII was increased in atria from AF patients compared to patients in sinus rhythm and from mice infused with Ang II compared with saline. Ang II treated mice had increased susceptibility to AF compared to saline treated WT mice, establishing Ang II as a risk factor for AF in mice. Knock in mice lacking critical oxidation sites in CaMKIIδ (MM-VV) and mice with myocardial-restricted transgenic over-expression of methionine sulfoxide reductase A (MsrA TG), an enzyme that reduces ox-CaMKII, were resistant to AF induction after Ang II infusion. Conclusions —Our studies suggest that CaMKII is a molecular signal that couples increased ROS with AF and that therapeutic strategies to decrease ox-CaMKII may prevent or reduce AF.Background— Atrial fibrillation (AF) is a growing public health problem without adequate therapies. Angiotensin II and reactive oxygen species are validated risk factors for AF in patients, but the molecular pathways connecting reactive oxygen species and AF are unknown. The Ca2+/calmodulin-dependent protein kinase II (CaMKII) has recently emerged as a reactive oxygen species–activated proarrhythmic signal, so we hypothesized that oxidized CaMKII&dgr; could contribute to AF. Methods and Results— We found that oxidized CaMKII was increased in atria from AF patients compared with patients in sinus rhythm and from mice infused with angiotensin II compared with mice infused with saline. Angiotensin II–treated mice had increased susceptibility to AF compared with saline-treated wild-type mice, establishing angiotensin II as a risk factor for AF in mice. Knock-in mice lacking critical oxidation sites in CaMKII&dgr; (MM-VV) and mice with myocardium-restricted transgenic overexpression of methionine sulfoxide reductase A, an enzyme that reduces oxidized CaMKII, were resistant to AF induction after angiotensin II infusion. Conclusions— Our studies suggest that CaMKII is a molecular signal that couples increased reactive oxygen species with AF and that therapeutic strategies to decrease oxidized CaMKII may prevent or reduce AF.

Crucial Role for Ca2+/Calmodulin-Dependent Protein Kinase-II in Regulating Diastolic Stress of Normal and Failing Hearts via Titin Phosphorylation

Rationale: Myocardial diastolic stiffness and cardiomyocyte passive force (Fpassive) depend in part on titin isoform composition and phosphorylation. Ca2+/calmodulin-dependent protein kinase-II (CaMKII) phosphorylates ion channels, Ca2+-handling proteins, and chromatin-modifying enzymes in the heart, but has not been known to target titin. Objective: To elucidate whether CaMKII phosphorylates titin and modulates Fpassive in normal and failing myocardium. Methods and Results: Titin phosphorylation was assessed in CaMKII&dgr;/&ggr; double-knockout (DKO) mouse, transgenic CaMKII&dgr;C-overexpressing mouse, and human hearts, by Pro-Q-Diamond/Sypro-Ruby staining, autoradiography, and immunoblotting using phosphoserine-specific titin-antibodies. CaMKII-dependent site-specific titin phosphorylation was quantified in vivo by mass spectrometry using stable isotope labeling by amino acids in cell culture mouse heart mixed with wild-type (WT) or DKO heart. Fpassive of single permeabilized cardiomyocytes was recorded before and after CaMKII-administration. All-titin phosphorylation was reduced by >50% in DKO but increased by up to ≈100% in transgenic versus WT hearts. Conserved CaMKII-dependent phosphosites were identified within the PEVK-domain of titin by quantitative mass spectrometry and confirmed in recombinant human PEVK-fragments. CaMKII also phosphorylated the cardiac titin N2B-unique sequence. Phosphorylation at specific PEVK/titin N2B-unique sequence sites was decreased in DKO and amplified in transgenic versus WT hearts. Fpassive was elevated in DKO and reduced in transgenic compared with WT cardiomyocytes. CaMKII-administration lowered Fpassive of WT and DKO cardiomyocytes, an effect blunted by titin antibody pretreatment. Human end-stage failing hearts revealed higher CaMKII expression/activity and phosphorylation at PEVK/titin N2B-unique sequence sites than nonfailing donor hearts. Table. Titin Phosphosites Downregulated in CaMKII&dgr;/&ggr; DKO vs WT Mouse Hearts, Identified by Quantitative Mass Spectrometry Conclusions: CaMKII phosphorylates the titin springs at conserved serines/threonines, thereby lowering Fpassive. Deranged CaMKII-dependent titin phosphorylation occurs in heart failure and contributes to altered diastolic stress.

Constitutively active phosphatase inhibitor-1 improves cardiac contractility in young mice but is deleterious after catecholaminergic stress and with aging

Phosphatase inhibitor-1 (I-1) is a distal amplifier element of beta-adrenergic signaling that functions by preventing dephosphorylation of downstream targets. I-1 is downregulated in human failing hearts, while overexpression of a constitutively active mutant form (I-1c) reverses contractile dysfunction in mouse failing hearts, suggesting that I-1c may be a candidate for gene therapy. We generated mice with conditional cardiomyocyte-restricted expression of I-1c (referred to herein as dTGI-1c mice) on an I-1-deficient background. Young adult dTGI-1c mice exhibited enhanced cardiac contractility but exaggerated contractile dysfunction and ventricular dilation upon catecholamine infusion. Telemetric ECG recordings revealed typical catecholamine-induced ventricular tachycardia and sudden death. Doxycycline feeding switched off expression of cardiomyocyte-restricted I-1c and reversed all abnormalities. Hearts from dTGI-1c mice showed hyperphosphorylation of phospholamban and the ryanodine receptor, and this was associated with an increased number of catecholamine-induced Ca2+ sparks in isolated myocytes. Aged dTGI-1c mice spontaneously developed a cardiomyopathic phenotype. These data were confirmed in a second independent transgenic mouse line, expressing a full-length I-1 mutant that could not be phosphorylated and thereby inactivated by PKC-alpha (I-1S67A). In conclusion, conditional expression of I-1c or I-1S67A enhanced steady-state phosphorylation of 2 key Ca2+-regulating sarcoplasmic reticulum enzymes. This was associated with increased contractile function in young animals but also with arrhythmias and cardiomyopathy after adrenergic stress and with aging. These data should be considered in the development of novel therapies for heart failure.