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Despite the clear association between myocardial injury, heart failure and depressed myocardial energetics, little is known about upstream signals responsible for remodeling myocardial metabolism after pathological stress. We found increased mitochondrial calmodulin kinase II (CaMKII) activation and left ventricular dilation in mice one week after myocardial infarction (MI) surgery. In contrast, mice with genetic mitochondrial CaMKII inhibition were protected from left ventricular dilation and dysfunction after MI. Mice with myocardial and mitochondrial CaMKII over-expression (mtCaMKII) had severe dilated cardiomyopathy and decreased ATP that caused elevated cytoplasmic resting (diastolic) Ca2+concentration and reduced mechanical performance. We mapped a metabolic pathway that allowed us to rescue disease phenotypes in mtCaMKII mice, providing new insights into physiological and pathological metabolic consequences of CaMKII signaling in mitochondria. Our findings suggest myocardial dilation, a disease phenotype lacking specific therapies, can be prevented by targeted replacement of mitochondrial creatine kinase, or mitochondrial-targeted CaMKII inhibition.

The multifunctional Ca 2+ /calmodulin-dependent kinase II (CaMKII) is activated by vasoconstrictors in vascular smooth muscle cells (VSMC), but its impact on vasoconstriction remains unknown. We hypothesized that CaMKII inhibition in VSMC decreases vasoconstriction. Using novel transgenic mice that express the inhibitor peptide CaMKIIN in smooth muscle (TG SM-CaMKIIN), we investigated the effect of CaMKII inhibition on L-type Ca 2+ channel current ( I Ca ), cytoplasmic and sarcoplasmic reticulum Ca 2+ , and vasoconstriction in mesenteric arteries. In mesenteric VSMC, CaMKII inhibition significantly reduced action potential duration and the residual I Ca 50 ms after peak amplitude, indicative of loss of L-type Ca 2+ channel–dependent I Ca facilitation. Treatment with angiotensin II or phenylephrine increased the intracellular Ca 2+ concentration in wild-type but not TG SM-CaMKIIN VSMC. The difference in intracellular Ca 2+ concentration was abolished by pretreatment with nifedipine, an L-type Ca 2+ channel antagonist. In TG SM-CaMKIIN VSMC, the total sarcoplasmic reticulum Ca 2+ content was reduced as a result of diminished sarcoplasmic reticulum Ca 2+ ATPase activity via impaired derepression of the sarcoplasmic reticulum Ca 2+ ATPase inhibitor phospholamban. Despite the differences in intracellular Ca 2+ concentration, CaMKII inhibition did not alter myogenic tone or vasoconstriction of mesenteric arteries in response to KCl, angiotensin II, and phenylephrine. However, it increased myosin light chain kinase activity. These data suggest that CaMKII activity maintains intracellular calcium homeostasis but is not required for vasoconstriction of mesenteric arteries.

Rationale: The sodium–calcium exchanger 1 (NCX1) is predominantly expressed in the heart and is implicated in controlling automaticity in isolated sinoatrial node (SAN) pacemaker cells, but the potential role of NCX1 in determining heart rate in vivo is unknown. Objective: To determine the role of Ncx1 in heart rate. Methods and Results: We used global myocardial and SAN-targeted conditional Ncx1 knockout ( Ncx1 −/− ) mice to measure the effect of the NCX current on pacemaking activity in vivo, ex vivo, and in isolated SAN cells. We induced conditional Ncx1 −/− using a Cre/loxP system. Unexpectedly, in vivo and ex vivo hearts and isolated SAN cells showed that basal rates in Ncx1 −/− (retaining ≈20% of control level NCX current) and control mice were similar, suggesting that physiological NCX1 expression is not required for determining resting heart rate. However, increases in heart rate and SAN cell automaticity in response to isoproterenol or the dihydropyridine Ca 2+ channel agonist BayK8644 were significantly blunted or eliminated in Ncx1 −/− mice, indicating that NCX1 is important for fight or flight heart rate responses. In contrast, the pacemaker current and L-type Ca 2+ currents were equivalent in control and Ncx1 −/− SAN cells under resting and isoproterenol-stimulated conditions. Ivabradine, a pacemaker current antagonist with clinical efficacy, reduced basal SAN cell automaticity similarly in control and Ncx1 −/− mice. However, ivabradine decreased automaticity in SAN cells isolated from Ncx1 −/− mice more effectively than in control SAN cells after isoproterenol, suggesting that the importance of NCX current in fight or flight rate increases is enhanced after pacemaker current inhibition. Conclusions: Physiological Ncx1 expression is required for increasing sinus rates in vivo, ex vivo, and in isolated SAN cells, but not for maintaining resting heart rate.

Myocardial cell death is initiated by excessive mitochondrial Ca(2+) entry causing Ca(2+) overload, mitochondrial permeability transition pore (mPTP) opening and dissipation of the mitochondrial inner membrane potential (ΔΨm). However, the signalling pathways that control mitochondrial Ca(2+) entry through the inner membrane mitochondrial Ca(2+) uniporter (MCU) are not known. The multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is activated in ischaemia reperfusion, myocardial infarction and neurohumoral injury, common causes of myocardial death and heart failure; these findings suggest that CaMKII could couple disease stress to mitochondrial injury. Here we show that CaMKII promotes mPTP opening and myocardial death by increasing MCU current (I(MCU)). Mitochondrial-targeted CaMKII inhibitory protein or cyclosporin A, an mPTP antagonist with clinical efficacy in ischaemia reperfusion injury, equivalently prevent mPTP opening, ΔΨm deterioration and diminish mitochondrial disruption and programmed cell death in response to ischaemia reperfusion injury. Mice with myocardial and mitochondrial-targeted CaMKII inhibition have reduced I(MCU) and are resistant to ischaemia reperfusion injury, myocardial infarction and neurohumoral injury, suggesting that pathological actions of CaMKII are substantially mediated by increasing I(MCU). Our findings identify CaMKII activity as a central mechanism for mitochondrial Ca(2+) entry in myocardial cell death, and indicate that mitochondrial-targeted CaMKII inhibition could prevent or reduce myocardial death and heart failure in response to common experimental forms of pathophysiological stress.

Sinus node dysfunction (SND) is a major public health problem that is associated with sudden cardiac death and requires surgical implantation of artificial pacemakers. However, little is known about the molecular and cellular mechanisms that cause SND. Most SND occurs in the setting of heart failure and hypertension, conditions that are marked by elevated circulating angiotensin II (Ang II) and increased oxidant stress. Here, we show that oxidized calmodulin kinase II (ox-CaMKII) is a biomarker for SND in patients and dogs and a disease determinant in mice. In wild-type mice, Ang II infusion caused sinoatrial nodal (SAN) cell oxidation by activating NADPH oxidase, leading to increased ox-CaMKII, SAN cell apoptosis, and SND. p47–/– mice lacking functional NADPH oxidase and mice with myocardial or SAN-targeted CaMKII inhibition were highly resistant to SAN apoptosis and SND, suggesting that ox-CaMKII–triggered SAN cell death contributed to SND. We developed a computational model of the sinoatrial node that showed that a loss of SAN cells below a critical threshold caused SND by preventing normal impulse formation and propagation. These data provide novel molecular and mechanistic information to understand SND and suggest that targeted CaMKII inhibition may be useful for preventing SND in high-risk patients.

Background— Catecholamines increase heart rate by augmenting the cAMP-responsive hyperpolarization-activated cyclic nucleotide-gated channel 4 pacemaker current ( I f ) and by promoting inward Na + /Ca 2+ exchanger current ( I NCX ) by a “Ca 2+ clock” mechanism in sinoatrial nodal cells (SANCs). The importance, identity, and function of signals that connect I f and Ca 2+ clock mechanisms are uncertain and controversial, but the multifunctional Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is required for physiological heart rate responses to β-adrenergic receptor (β-AR) stimulation. The aim of this study was to measure the contribution of the Ca 2+ clock and CaMKII to cardiac pacing independent of β-AR agonist stimulation. Methods and Results— We used the L-type Ca 2+ channel agonist Bay K8644 (BayK) to activate the SANC Ca 2+ clock. BayK and isoproterenol were similarly effective in increasing rates in SANCs and Langendorff-perfused hearts from wild-type control mice. In contrast, SANCs and isolated hearts from mice with CaMKII inhibition by transgenic expression of an inhibitory peptide (AC3-I) were resistant to rate increases by BayK. BayK only activated CaMKII in control SANCs but increased L-type Ca 2+ current ( I Ca ) equally in all SANCs, indicating that increasing I Ca was insufficient and suggesting that CaMKII activation was required for heart rate increases by BayK. BayK did not increase I f or protein kinase A-dependent phosphorylation of phospholamban (at Ser16), indicating that increased SANC Ca 2+ by BayK did not augment cAMP/protein kinase A signaling at these targets. Late-diastolic intracellular Ca 2+ release and I NCX were significantly reduced in AC3-I SANCs, and the response to BayK was eliminated by ryanodine in all groups. Conclusions— The Ca 2+ clock is capable of supporting physiological fight-or-flight responses, independent of β-AR stimulation or I f increases. Complete Ca 2+ clock and β-AR stimulation responses require CaMKII.

Myocardial mitochondrial Ca(2+) entry enables physiological stress responses but in excess promotes injury and death. However, tissue-specific in vivo systems for testing the role of mitochondrial Ca(2+) are lacking. We developed a mouse model with myocardial delimited transgenic expression of a dominant negative (DN) form of the mitochondrial Ca(2+) uniporter (MCU). DN-MCU mice lack MCU-mediated mitochondrial Ca(2+) entry in myocardium, but, surprisingly, isolated perfused hearts exhibited higher O2 consumption rates (OCR) and impaired pacing induced mechanical performance compared with wild-type (WT) littermate controls. In contrast, OCR in DN-MCU-permeabilized myocardial fibers or isolated mitochondria in low Ca(2+) were not increased compared with WT, suggesting that DN-MCU expression increased OCR by enhanced energetic demands related to extramitochondrial Ca(2+) homeostasis. Consistent with this, we found that DN-MCU ventricular cardiomyocytes exhibited elevated cytoplasmic [Ca(2+)] that was partially reversed by ATP dialysis, suggesting that metabolic defects arising from loss of MCU function impaired physiological intracellular Ca(2+) homeostasis. Mitochondrial Ca(2+) overload is thought to dissipate the inner mitochondrial membrane potential (ΔΨm) and enhance formation of reactive oxygen species (ROS) as a consequence of ischemia-reperfusion injury. Our data show that DN-MCU hearts had preserved ΔΨm and reduced ROS during ischemia reperfusion but were not protected from myocardial death compared with WT. Taken together, our findings show that chronic myocardial MCU inhibition leads to previously unanticipated compensatory changes that affect cytoplasmic Ca(2+) homeostasis, reprogram transcription, increase OCR, reduce performance, and prevent anticipated therapeutic responses to ischemia-reperfusion injury.

Inhibition of the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) or depletion of sarcoplasmic reticulum (SR) Ca2+ stores protects against apoptosis from excessive isoproterenol (Iso) stimulation in cultured ventricular myocytes, suggesting that CaMKII inhibition could be a novel approach to reducing cell death in conditions of increased adrenergic tone, such as myocardial infarction (MI), in vivo. We used mice with genetic myocardial CaMKII inhibition due to transgenic expression of a highly specific CaMKII inhibitory peptide (AC3-I) to test whether CaMKII was important for apoptosis in vivo. A second line of mice expressed a scrambled, inactive form of AC3-I (AC3-C). AC3-C and wild-type (WT) littermates were used as controls. AC3-I mice have reduced SR Ca2+ content and are resistant to Iso- and MI-induced apoptosis compared with AC3-C and WT mice. Phospholamban (PLN) is a target for modulation of SR Ca2+ content by CaMKII. PLN−/− mice have increased susceptibility to Iso-induced apoptosis. Verapamil pretreatment prevented Iso-induced apoptosis in PLN−/− mice, indicating the involvement of a Ca2+-dependent pathway. AC3-I and AC3-C mice were bred into a PLN−/− background. Loss of PLN increased and equalized SR Ca2+ content in AC3-I, AC3-C, and WT mice and abolished the resistance to apoptosis in AC3-I mice after MI. There was a trend ( P = 0.07) for increased Iso-induced apoptosis in AC3-I mice lacking PLN compared with AC3-I mice with PLN. These findings indicate CaMKII is proapoptotic in vivo and suggest that regulation of SR Ca2+ content by PLN contributes to the antiapoptotic mechanism of CaMKII inhibition.

Heart rate increases are a fundamental adaptation to physiological stress, while inappropriate heart rate increases are resistant to current therapies. However, the metabolic mechanisms driving heart rate acceleration in cardiac pacemaker cells remain incompletely understood. The mitochondrial calcium uniporter (MCU) facilitates calcium entry into the mitochondrial matrix to stimulate metabolism. We developed mice with myocardial MCU inhibition by transgenic expression of a dominant-negative (DN) MCU. Here, we show that DN-MCU mice had normal resting heart rates but were incapable of physiological fight or flight heart rate acceleration. We found that MCU function was essential for rapidly increasing mitochondrial calcium in pacemaker cells and that MCU-enhanced oxidative phoshorylation was required to accelerate reloading of an intracellular calcium compartment before each heartbeat. Our findings show that MCU is necessary for complete physiological heart rate acceleration and suggest that MCU inhibition could reduce inappropriate heart rate increases without affecting resting heart rate.

Diabetes, a worldwide epidemic, represents a major public health problem and the vast majority of the patients with diabetes presents with insulin resistance, a fundamental manifestation of this disease. Insulin resistance impairs glucose uptake into skeletal muscle, which takes up about 80% of postprandial glucose in healthy individuals (1). Thus, skeletal muscle plays an indispensable role in maintaining glucose homeostasis. However, the mechanisms underlying the development of insulin resistance remains poorly understood, which perhaps accounts for the lack of effective therapies. Mitochondria, where low levels of superoxide radicals are constitutively generated as a by-product of electron transport, serve as the powerhouse and are also considered a main source for overproduction of reactive oxygen species (ROS) triggered by diabetes (2,3). Oxidative stress is a key pathological signal leading to diabetes complications (4,5). It has been disappointing that broad-spectrum antioxidant therapies have not been effective in improving outcomes in high-risk patients, including patients with diabetes (6), when used as primary prevention, suggesting that detailed knowledge of oxidative injury mechanisms is necessary to develop …

Excessive activation of the beta-adrenergic, angiotensin II (Ang II) and aldosterone signaling pathways promotes mortality after myocardial infarction, and antagonists targeting these pathways are core therapies for treating this condition. Catecholamines and Ang II activate the multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), the inhibition of which prevents isoproterenol-mediated and Ang II-mediated cardiomyopathy. Here we show that aldosterone exerts direct toxic actions on myocardium by oxidative activation of CaMKII, causing cardiac rupture and increased mortality in mice after myocardial infarction. Aldosterone induces CaMKII oxidation by recruiting NADPH oxidase, and this oxidized and activated CaMKII promotes matrix metalloproteinase 9 (MMP9) expression in cardiomyocytes. Myocardial CaMKII inhibition, overexpression of methionine sulfoxide reductase A (an enzyme that reduces oxidized CaMKII) or NADPH oxidase deficiency prevented aldosterone-enhanced cardiac rupture after myocardial infarction. These findings show that oxidized myocardial CaMKII mediates the cardiotoxic effects of aldosterone on the cardiac matrix and establish CaMKII as a nodal signal for the neurohumoral pathways associated with poor outcomes after myocardial infarction.

Excessive activation of calmodulin kinase II (CaMKII) causes arrhythmias and heart failure, but the cellular mechanisms for CaMKII-targeted proteins causing disordered cell membrane excitability and myocardial dysfunction remain uncertain. Failing human cardiomyocytes exhibit increased CaMKII and voltage-gated Ca 2+ channel (Ca V 1.2) activity, and enhanced expression of a specific Ca V 1.2 β-subunit protein isoform ( β 2a ). We recently identified Ca V 1.2 β 2a residues critical for CaMKII phosphorylation (Thr 498) and binding (Leu 493), suggesting the hypothesis that these amino acids are crucial for cardiomyopathic consequences of CaMKII signaling. Here we show WT β 2a expression causes cellular Ca 2+ overload, arrhythmia-triggering cell membrane potential oscillations called early afterdepolarizations (EADs), and premature death in paced adult rabbit ventricular myocytes. Prevention of intracellular Ca 2+ release by ryanodine or global cellular CaMKII inhibition reduced EADs and improved cell survival to control levels in WT β 2a -expressing ventricular myocytes. In contrast, expression of β 2a T498A or L493A mutants mimicked the protective effects of ryanodine or global cellular CaMKII inhibition by reducing Ca 2+ entry through Ca V 1.2 and inhibiting EADs. Furthermore, Ca V 1.2 currents recorded from cells overexpressing CaMKII phosphorylation- or binding-incompetent β 2a subunits were incapable of entering a CaMKII-dependent high-activity gating mode (mode 2), indicating that β 2a Thr 498 and Leu 493 are required for Ca V 1.2 activation by CaMKII in native cells. These data show that CaMKII binding and phosphorylation sites on β 2a are concise but pivotal components of a molecular and biophysical and mechanism for EADs and impaired survival in adult cardiomyocytes.

Increasing evidence suggests that cardiac pacemaking is the result of two sinoatrial node (SAN) cell mechanisms: a 'voltage clock' and a Ca(2+) dependent process, or 'Ca(2+) clock.' The voltage clock initiates action potentials (APs) by SAN cell membrane potential depolarization from inward currents, of which the pacemaker current (I(f)) is thought to be particularly important. A Ca(2+) dependent process triggers APs when sarcoplasmic reticulum (SR) Ca(2+) release activates inward current carried by the forward mode of the electrogenic Na(+)/Ca(2+) exchanger (NCX). However, these mechanisms have mostly been defined in rodents or rabbits, but are unexplored in single SAN cells from larger animals. Here, we used patch-clamp and confocal microscope techniques to explore the roles of the voltage and Ca(2+) clock mechanisms in canine SAN pacemaker cells. We found that ZD7288, a selective I(f) antagonist, significantly reduced basal automaticity and induced irregular, arrhythmia-like activity in canine SAN cells. In addition, ZD7288 impaired but did not eliminate the SAN cell rate acceleration by isoproterenol. In contrast, ryanodine significantly reduced the SAN cell acceleration by isoproterenol, while ryanodine reduction of basal automaticity was modest ( approximately 14%) and did not reach statistical significance. Importantly, pretreatment with ryanodine eliminated SR Ca(2+) release, but did not affect basal or isoproterenol-enhanced I(f). Taken together, these results indicate that voltage and Ca(2+) dependent automaticity mechanisms coexist in canine SAN cells, and suggest that I(f) and SR Ca(2+) release cooperate to determine baseline and catecholamine-dependent automaticity in isolated dog SAN cells.

SummaryCalcium/calmodulin (Ca2+/CaM)-dependent protein kinase II (CaMKII) couples increases in cellular Ca2+ to fundamental responses in excitable cells. CaMKII was identified over 20 years ago by activation dependence on Ca2+/CaM, but recent evidence shows that CaMKII activity is also enhanced by pro-oxidant conditions. Here we show that oxidation of paired regulatory domain methionine residues sustains CaMKII activity in the absence of Ca2+/CaM. CaMKII is activated by angiotensin II (AngII)-induced oxidation, leading to apoptosis in cardiomyocytes both in vitro and in vivo. CaMKII oxidation is reversed by methionine sulfoxide reductase A (MsrA), and MsrA−/− mice show exaggerated CaMKII oxidation and myocardial apoptosis, impaired cardiac function, and increased mortality after myocardial infarction. Our data demonstrate a dynamic mechanism for CaMKII activation by oxidation and highlight the critical importance of oxidation-dependent CaMKII activation to AngII and ischemic myocardial apoptosis.