Background— Impairment of intracellular Ca 2+ homeostasis and mitochondrial function has been implicated in the development of cardiomyopathy. Mitochondrial Ca 2+ uptake is thought to be mediated by the Ca 2+ uniporter (MCU) and a thus far speculative non-MCU pathway. However, the identity and properties of these pathways are a matter of intense debate, and possible functional alterations in diseased states have remained elusive. Methods and Results— By patch clamping the inner membrane of mitochondria from nonfailing and failing human hearts, we have identified 2 previously unknown Ca 2+ -selective channels, referred to as mCa1 and mCa2. Both channels are voltage dependent but differ significantly in gating parameters. Compared with mCa2 channels, mCa1 channels exhibit a higher single-channel amplitude, shorter openings, a lower open probability, and 3 to 5 subconductance states. Similar to the MCU, mCa1 is inhibited by 200 nmol/L ruthenium 360, whereas mCa2 is insensitive to 200 nmol/L ruthenium 360 and reduced only by very high concentrations (10 μmol/L). Both mitochondrial Ca 2+ channels are unaffected by blockers of other possibly Ca 2+ -conducting mitochondrial pores but were activated by spermine (1 mmol/L). Notably, activity of mCa1 and mCa2 channels is decreased in failing compared with nonfailing heart conditions, making them less effective for Ca 2+ uptake and likely Ca 2+ -induced metabolism. Conclusions— Thus, we conclude that the human mitochondrial Ca 2+ uptake is mediated by these 2 distinct Ca 2+ channels, which are functionally impaired in heart failure. Current properties reveal that the mCa1 channel underlies the human MCU and that the mCa2 channel is responsible for the ruthenium red–insensitive/low-sensitivity non-MCU–type mitochondrial Ca 2+ uptake.

L-Type calcium channels are multiprotein complexes composed of pore-forming (Ca V 1.2) and modulatory auxiliary ␣ 2 ␦-and ␤-subunits. We demonstrate expression of two different isoforms for the ␤ 2 -subunit (␤ 2a , ␤ 2b ) and the ␤ 3 -subunit (␤ 3a , ␤ 3trunc ) in human non-failing and failing ischemic myocardium. Quantitatively, in the left ventricle expression of ␤ 2b transcripts prevails in the order of > ␤ 3 > > ␤ 2a . The expressed cardiac full-length ␤ 3 -subunit is identical to the ␤ 3a -isoform, and ␤ 3trunc results from deletion of exon 6 (20 nn) entailing a reading frameshift and translation stop at nucleotide position 495. In failing ischemic myocardium ␤ 3trunc expression increases whereas overall ␤ 3 expression remains unchanged. Heterologous coexpression studies demonstrated that ␤ 2 induced larger currents through rabbit and human cardiac Ca V 1.2 pore subunits than ␤ 3 isoforms. All ␤-subunits increased channel availability at single channel level, but ␤ 2 exerted an additional, marked stimulation of rapid gating (open and closed times, first latency), leading to higher peak current values. We conclude that cardiac ␤-subunit isoforms differentially modulate calcium inward currents because of regulatory effects within the channel protein complex. Moreover, differences in the various ␤-subunit gene products present in human heart might account for altered single channel behavior found in human heart failure.

Background-The pacemaker current I f is present in atrial and ventricular myocytes. However, it remains controversial whether I f overexpression in diseased states might play a role for arrhythmogenesis, because first I f activation in whole-cell recordings hardly overlapped the diastolic voltage of working myocardium.

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BackgroundIncreased activity of single ventricular L-type Ca2+-channels (L-VDCC) is a hallmark in human heart failure. Recent findings suggest differential modulation by several auxiliary β-subunits as a possible explanation.Methods and ResultsBy molecular and functional analyses of human and murine ventricles, we find that enhanced L-VDCC activity is accompanied by altered expression pattern of auxiliary L-VDCC β-subunit gene products. In HEK293-cells we show differential modulation of single L-VDCC activity by coexpression of several human cardiac β-subunits: Unlike β1 or β3 isoforms, β2a and β2b induce a high-activity channel behavior typical of failing myocytes. In accordance, β2-subunit mRNA and protein are up-regulated in failing human myocardium. In a model of heart failure we find that mice overexpressing the human cardiac CaV1.2 also reveal increased single-channel activity and sarcolemmal β2 expression when entering into the maladaptive stage of heart failure. Interestingly, these animals, when still young and non-failing (“Adaptive Phase”), reveal the opposite phenotype, viz : reduced single-channel activity accompanied by lowered β2 expression. Additional evidence for the cause-effect relationship between β2-subunit expression and single L-VDCC activity is provided by newly engineered, double-transgenic mice bearing both constitutive CaV1.2 and inducible β2 cardiac overexpression. Here in non-failing hearts induction of β2-subunit overexpression mimicked the increase of single L-VDCC activity observed in murine and human chronic heart failure.ConclusionsOur study presents evidence of the pathobiochemical relevance of β2-subunits for the electrophysiological phenotype of cardiac L-VDCC and thus provides an explanation for the single L-VDCC gating observed in human and murine heart failure.